Genome-wide profiling of RNA 2’-O-methylation in neurons and identification of orphan snoRNA targets

Genome-wide profiling of RNA 2’-O-methylation in neurons and identification of orphan snoRNA targets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genome-wide profiling of RNA 2’-O-methylation in neurons and identification of orphan snoRNA targets Gordon Carmichael, Xuan Ye, Yaling Liu, Yinzhou Zhu, Saran Alshawi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8523796/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract We have compared genome-wide patterns of RNA 2’- O -methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2’- O -methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders. Biological sciences/Neuroscience/Molecular neuroscience Biological sciences/Molecular biology/Transcriptomics Biological sciences/Molecular biology/RNA metabolism/RNA modification Biological sciences/Molecular biology/Non-coding RNAs/Small RNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction To date, over 170 distinct RNA modifications have been identified and characterized 1 – 3 . These modifications, which don't alter RNA sequence, play important roles in regulating gene expression and various physiological and pathological processes. Among these RNA modifications, 2’- O -methylation (Nm) is a widespread RNA modification found in multiple RNA types and species, 4 and it is among the most abundant in the cell owing to numerous sites on rRNAs and small RNAs. 2’- O -methylation in RNA is thought to primarily function to increase RNA stability, promote RNA folding, facilitate protein binding, and make RNA less susceptible to hydrolysis and cleavage 5 . The functional impact of Nm is most evident in the regulation of the translational apparatus 6 . Differential rRNA methylation patterns have been shown to modulate ribosome heterogeneity, effectively "tuning" translation to meet specific cellular demands 7 – 11 . Beyond the ribosome, Nm occurs in mRNA, viral RNA, and small RNAs, where it influences alternative splicing, mRNA export, and translational fidelity 12 – 20 . Notably, Nm can sterically hinder the association of RNA-binding proteins (RBPs) and disrupt long-range tertiary interactions that rely on 2’-OH hydrogen bonding or divalent metal ion coordination 21 and the presence of a 2’-O-methyl group can sterically hinder RNA-binding protein association 22 , 23 . The majority of cellular Nm is directed by box C/D snoRNAs, which function as antisense guides within a ribonucleoprotein (snoRNP) complex. Box C/D snoRNAs are defined by conserved sequence motifs—boxes C/C’ (RUGAUGA) and D/D’ (CUGA). The snoRNA utilizes 10–15 nucleotide antisense elements (ASEs) to base-pair with target transcripts. Canonically, the target nucleotide is positioned exactly five nucleotides upstream of the D or D’ box. The snoRNA scaffolds a core protein tetramer consisting of Fibrillarin (the methyltransferase), NOP56, NOP58, and SNU13. While snoRNA-mediated methylation is the dominant pathway, independent mechanisms exist, such as the FTSJ3-mediated methylation of the HIV-1 genome, though these non-canonical pathways remain under-characterized 16 . In humans, 137 out of 267 annotated box C/D snoRNAs 24 , 25 have no specified target and are therefore classified as orphan snoRNAs. Possible functions of orphan snoRNAs have been recently reviewed 26 . A number of snoRNAs have been shown to target specific sites on mRNA 15 , 27 and even tRNA 28 . Also, some have been reported to be involved in alternative splicing 29 , alternative polyadenylation 30 and other noncanonical processes 31 – 33 . Greater in-depth exploration of Nm biological functions has been limited due to the lack of robust screening tools to detect Nm sites. Mapping Nm positions has proven challenging for many reasons, including resistance to hydrolysis 5 , the lack of an antibody that can detect Nm and a possibly low stoichiometry of Nm on mRNA. Several high-throughput Nm detection methods have been developed in recent years, including 2’- O Me-seq 34 , RiboMeth-seq 35–37 , RibOxi-seq 38 , 39 , Nm-seq 12 , Nm-mut-seq 17 and NJU-seq 19 . RiboMeth-seq has emerged as the only method that consistently detects all known Nm sites in human rRNAs 35 – 37 . Most importantly, RiboMeth-seq is the only currently available method for the quantitative evaluation of Nm levels at multiple positions, thus providing information on potential Nm variations and providing Nm profiles. However, RiboMeth-seq cannot be used to identify mRNA sites. Recently, Oxford Nanopore Technologies Direct RNA Sequencing (DRS) has been employed by several groups to investigate transcriptome-wide Nm profiles 40 , 41 . However, Nanopore-based Nm detection is complicated by signal changes that include read mismatches, deletions, and mutations at neighboring positions. As a result, accurate determination of Nm locations using Nanopore DRS often requires machine learning–based computational analysis and/or comparison with snoRNA knockout conditions. We have developed RibOxi-seq, which allows the detection of Nm even from small amounts of RNA, including mRNA and non-coding RNA 38 , 39 . Like Nm-seq, this method includes multiple iterative cycles of oxidation/beta elimination-dephosphorylation reactions to sequentially eliminate unmodified nucleotides from 3’ ends but leave the Nm sites intact. Finally, only RNA fragments with 2’- O -methylated 3’ ends can be ligated to linkers for library preparation (Fig. 1 a). Consequently, Nm sites generate a positive signal and not a lack of signal, in contrast to the methods based on the inhibition of reverse transcription (RT) reactions or resistance to hydrolysis. In this study we have further optimized the RibOxi-seq method (now RibOxi-seq2) to make it more sensitive and reproducible. Validating specific Nm positions has also proved challenging. One sensitive method is RTL-P 42 . When reverse transcription (RT) is performed with low concentrations of dNTPs, the RT enzyme has difficulty incorporating nucleotides opposite a 2’- O -methylated residue. This leads to premature termination or pausing of cDNA synthesis one nucleotide before the methylated site. However, this method is not reliable for low-abundance RNAs and some structured RNAs 27 . Recently, a site-specific Nm quantification tool, Nm-VAQ, was described 19 . The Nm-VAQ (2’-O-methylation Validation and Absolute Quantification) method can validate the presence and precisely quantify the stoichiometry of Nm at specific RNA nucleotides by leveraging the property that 2’- O -methylated RNA is resistant to cleavage by RNase H when it is part of an RNA/DNA hybrid duplex. Several box C/D snoRNAs have been linked to neurodevelopmental disorders. One of the most notable examples is the orphan C/D box snoRNA cluster SNORD116, which is implicated in Prader–Willi Syndrome (PWS), a rare imprinting disorder affecting approximately 1 in 15,000 newborns 43 – 45 . PWS results from disruptions of the paternally inherited chromosome 15q11–q13 region, which harbors ~ 30 tandemly arranged SNORD116 genes 46 – 49 . This locus is subject to genomic imprinting, with expression dependent on the paternal allele, and deletions in this region are sufficient to cause the disease. Another imprinted snoRNA cluster with neurological relevance is located on chromosome 14q32.2, where the maternally imprinted SNORD112/113/114 genes reside. Deletions affecting this region give rise to Kagami–Ogata Syndrome, a rare multisystem disorder characterized by skeletal abnormalities and developmental delays including neurodevelopmenlal defects 50 , 51 . To date, there has been no study published on genome-wide Nm profiling in neurons, or in cell models of PWS. To address these knowledge gaps, we have used RibOxi-seq2 to examine isogenic pairs of neurons that only differ in the expression of several orphan box C/D clusters. This allowed us to not only identify neuronal sites of rRNA and small RNA modification but also thousands of mRNA sites, some of which appear to be direct targets of orphan snoRNAs. Results Human embryonic stem cell lines H9 and CT2 were engineered to harbor a doxycycline-inducible neurogenin 2 (NGN2) gene that allows rapid and reproducible differentiation into early postnatal forebrain cortical neurons. Separate isogenic lines were also created in which a small deletion (smDEL) was introduced only on the paternal chromosome 15, removing a cluster of 30 related box C/D snoRNAs (the SNORD116 cluster) that has been implicated in the pathology of Prader-Willi syndrome 45 . The generation of these cells was recently described 52 and NGN2-induced neurons (iNs) from these cell lines, H9, H9-smDEL, CT2, and CT2-smDEL, have been extensively characterized at the transcriptomic level 53 . Apart from a modest number of overall transcriptomic differences, there are two key features that differentiate the WT and smDEL iNs. First, both the H9-smDEL and CT2-smDEL iNs lack expression of the paternally imprinted SNORD116 cluster of orphan snoRNAs that have been postulated to direct Nms on so far unidentified targets (Fig. 1 b,c). Second, although originally derived from parent CT2 cells, the CT2-smDEL cells also differ in their expression of a maternally imprinted locus on chromosome 14q32 54 and thus differ from CT2 iNs in the expression of the additional orphan SNORD113/114 clusters, comprising 9 and 31 copies, respectively ( Fig. 1 c ) . Importantly, both SNORD113/114 and SNORD116 snoRNAs are expressed in the brain. Thus, the CT2-smDEL iNs can serve as useful knockout lines for several specific and physiologically relevant subsets of orphan snoRNAs. Ribosomal RNA modifications in stem cells and neurons . Given that the relationship between Nm and neuronal disorders remains poorly understood, we sought to gain detailed insights into Nm patterns in an isogenic pair of neuronal populations. RNAs were subjected to RibOxi-seq2 and sites displayed on the UCSC Genome Browser. Figure 2 a-f shows representative profiles of 18S and 28S rRNA for CT2, CT2-smDEL, H9, and H9-smDEL iNs. Every peak observed represents an annotated or validated Nm site ( Supplementary Data 1 ). Notably, the peaks are qualitatively consistent between the four cell samples, revealing not only the reproducibility of the RibOxi-seq2 method but also reinforcing the fact that SNORD116 snoRNAs are true orphans as they appear not to target any rRNA Nm. An alternative method, RiboMeth-seq, was next used to corroborate and extend these results (Fig. 2 c-f). This method has been shown to be highly sensitive and quantitative for rRNA Nm mapping. RiboMeth-seq identified 44 Nm sites in 18S rRNA (3 Nm sites were validated using the site-specific Nm quantification tool, Nm-VAQ 19 ) and 67 Nm sites in 28S rRNA, while RibOxi-seq2 data identified 42 Nm sites in 18S rRNA and 61 Nm sites in 28S rRNA ( Supplementary Data 1 ). In 18S rRNA, using both RibOxi-seq2 and RiboMeth-seq, we identified two Nm sites, Um966 and Um1445, that are known to be highly modified as pseudouridines (Ψ), suggesting the likelihood of double modification (Ψm) at these sites (for this cellular context) 9 . Likewise, in 28S rRNA, U3818 is known to be modified as Ψm3818, and we also see it as Nm in RibOxi-seq2 tracks and validated this by Nm-VAQ. One weakness of RibOxi-seq2 is that if there are consecutive Nm sites, only the most 3’ site is observed, and this weakness accounts for most of the discrepancy between the two methods. Thus, 61/63 detectable sites in 28S rRNA were observed by RibOxi-seq2. In order to compare rRNA modifications between pluripotent cells and neurons, we also performed RiboMeth-seq on undifferentiated H9 cells (Fig. 2 c,d and Supplementary Data 1 .) Nm profiles between H9 cells and H9 iNs were essentially the same except that 18S U354 is unmethylated in stem cells and largely methylated in iNs ( Fig. 2 c-f). These data reveal dynamic ribosomal RNA patterns across different cell lines, suggesting that specific modification levels may contribute to cell-type-specific differentiation. Interestingly, 18S Um354 has previously been associated with differentiation and is directed by SNORD90 55 . It was recently reported that SNORD113/114 snoRNAs may maintain hematopoietic stem cell self-renewal at least in part by altering rRNA Nm levels 56 . Here we observe no 18S or 28S rRNA Nm differences between CT2 and CT2 smDEL iNs, which differ in expression of SNORD113/114. Nm profiles were also analyzed in 5.8S rRNA species and no differences in Nm were detected. Small RNA modifications in neurons . RibOxi-seq revealed Nm sites in a number of tRNAs. For example, the first nucleotide in the anticodon loop was 2’-O-methylated in tRNA-Gly-CCC-2-2 (Um31), tRNA-Val-CAC-1-4 (Cm31), tRNA-Ala-AGC-2-1 (Um31) and tRNA-Cys-GCA-2-2 (Cm31) (Fig. 2 g and Supplementary Data 1 ). This position is highly conserved compared to the Nm at nucleotide 32 reported in tRNA-Phe, and has been shown to be enzymatically catalyzed by the protein FTSJ1 in human (yeast homolog Trm7), or mediated by SNORD97 or SNORD133 in a snoRNA-dependent fashion 28 . Since these Nm in the anticodon region were not mediated by SNORD113/114/116 snoRNAs, no differences were observed between H9/CT2 wild type and smDEL iNs. Further, Um38 in tRNA-Gly-CCC-2-2 and Cm39 in tRNA-Glu-CTC-1-7 were identified at the first nucleotide following the anticodon loop (Fig. 2 g and Supplementary Data 1 ). These modifications are conserved and correspond to previously annotated (Um/Gm/Ψm39) 57 . The Nm at position 39 was reported to be catalyzed by a snoRNP complex 58 . As expected, m5Um (2’-O-methyl-5-methyluridine) residues located at the first nucleotide in the T-loop region were also reproducibly captured, including tRNA-Lys-CTT-2-5 (m5Um-54), tRNA-Leu-AAG-2-4 (m5Um-63), tRNA-Gly-GCC-2-4 (m5Um-52), tRNA-Glu-CTC-1-7 (m5Um-53), and tRNA-Asn-GTT-1-1 (m5Um-55) (Fig. 2 g and Supplementary Data 1 ). The m5Um identity and position match previous mass spectrometry results 57 , indicating that the m5Um modification also resists sodium periodate treatment, and RibOxi-seq2 cannot clearly differentiate between Nm and m5Um modifications since both are 2’-O-methylated nucleotides. In addition, reproducible Nm sites were identified in U1 (Am70), U4 (Am65), U5 (Gm37, Um41, Cm45), and U6 (Am70, Cm77) snRNAs (Fig. 2 h ). The sites extend our recent work using RibOxi-seq to map Nm sites on U6, supporting the robustness of RibOxi-seq in mapping Nm even in small RNA species 59 . Nm modifications in U2 snRNA were not detected, most likely due to the RNA fragmentation procedure failing to enrich small U2 snRNA fragments. Interestingly, we found a novel Nm site in 7SK RNA, Gm240 ( Supplementary Data 1 ). This site is two nucleotides downstream of A238 which has been reported to be modified as m6A 60 . As no Nm sites have been reported previously in 7SK RNA, we don’t yet know whether this modification occurs in cells other than neurons. Collectively, the RibOxi-seq2 method faithfully identified Nm sites in rRNA, tRNA, and snRNA. As expected, no Nm level changes were found in small RNAs between wild type and smDEL iNs. Together, these results suggest that RibOxi-seq2 may prove useful for mapping many Nm sites even in small RNAs. Widespread and robust modifications in mRNAs . A recent study identified thousands of Nm sites in mRNAs of human and mouse cell lines using NJU-seq and validated a number of them using Nm-VAQ 19 . Of thousands of Nm sites identified, about a third were conserved between different cell types. These authors revealed a broad distribution of Nm sites on mRNAs and observed that in their system most validated Nm sites were methylated at ratios from 1% to 30%. Here using RibOxi-seq2 we have also identified thousands of likely Nm sites within mRNAs and lncRNAs, a number of which were validated by Nm-VAQ ( Supplementary Data 2 ). Importantly, this approach yielded consistent Nm peaks across H9, CT2, and smDEL cell lines, providing strong evidence for the robustness of the method (Fig. 3 a). Owing to a higher sequencing depth in the CT2 samples, we could identify more sites in the CT2 data (989 Nm sites with Nm score 1000 or higher) than in the H9 data (736 Nm sites with Nm score 1000 or higher). When we selected the top 500 sites between both cell types, it was clear that a majority of identified Nm sites are shared between the two parental cell lines (Fig. 3 b). Also, for both H9 and CT2 iNs, Nm profiles were almost identical between the WT and smDEL cells, again highlighting the reproducibility of RibOxi-seq2. Overall, about 40% of Nm sites were in 3’-UTRs, 5% in 5’-UTRs, and the rest in coding exons (Fig. 3 c). While most are shared, some Nm sites were observed only in H9 iNs or CT2 iNs. One example of this is shown in Fig. 3 d. UCHL1 exhibits a strong site in H9 iNs but not in CT2 iNs. Also, the major TOP1 Nm site is in exon 9 in H9 cells but in exon 12 in CT2 cells (Fig. 3 e). Gene ontology analysis revealed that Nm sites are broadly distributed in biological processes, with no clear preference for any pathway (not shown). Next, we employed Nm-VAQ to validate the modification levels at several mRNA Nm sites. Results are shown in Fig. 4 . Surprisingly, a number of neuronal mRNAs exhibited very high degrees of methylation, in some cases approaching complete modification (Fig. 4 f). For instance, two sites in ACTB exons 4 and 5 were methylated at 97% and 100% (Fig. 4 a), respectively (N.B., RibOxi-seq2 Nm browser peaks as displayed are not quantitative); a site in the 5′-UTR translational control element of TPT1 was modified at 88% (Fig. 4 c); and a site in SNCG exon 3 showed 100% modification (Fig. 4 d). In addition, we detected a site in NUDT21 exon 1 at 50% ( Supplementary Data 2 ) and another in the 3′-UTR of FAM171A1 at 43% (Fig. 4 b), indicating that not all sites are heavily modified. By contrast, certain Nm sites displayed much lower modification levels. For example, a site in MT-CO1 was modified at only 6.7% and one in FGF13 at 24% (Fig. 4 e and Supplementary Data 2 ). Taken together, these results demonstrate that, at least in iNs, mRNA Nm modifications occur across a wide dynamic range, from nearly absent to fully saturated. Additionally, the Nm sites we identified in neuronal cell lines did not overlap with the mRNA Nm sites previously reported in HeLa cells 19 , suggesting dynamic and/or cell line–specific Nm regulation and variation, or methodologic differences. Lack of canonical snoRNA targeting of most mRNA sites. While all rRNA and most snRNA Nm sites are known to be the targets of box C/D snoRNAs or scaRNAs, this has not been shown yet for most mRNA sites. Our large number of shared Nm sites from our four iN cell lines offered the possibility to test this possibility. We selected CT2 sites with Nm scores above 500 (over 500 sites) and analyzed them by SnoScan 61 for canonical complementarity to known snoRNAs. Thus, successful hits must exhibit significant complementarity to snoRNAs with the Nm site positioned 5 nucleotides upstream of the snoRNA box D or box D’ (CUGA) motif. Surprisingly, almost none of the abundant shared sites satisfied these criteria. Thus, overall, the vast majority of Nm sites on this list could not be assigned to canonical snoRNA interactions. However, some sites not shared between cells and of lower expression levels could be matched (see below). We next explored the possibility that noncanonical snoRNA-mRNA interactions may play a role in some Nm modifications. While the abundant U3, U8 and U13 snoRNAs have not been previously associated with methyltransferase activity, a recent report studying mRNA-snoRNA physical interactions (sno-KARR-seq) revealed that a high proportion of mRNAs apparently associated with U3 snoRNA, although not necessarily involving canonical basepairing 62 . Further, those authors reported that U3 snoRNA knockdown led to a significant decrease in overall cellular Nm levels. Another group investigating crosslinking of snoRNA-mRNA hybrids also noted a very high number of U3-mRNA interactions 63 . Together, these studies raised the possibility that this snoRNA may have a role in the cell other than in rRNA processing. Interestingly, the sequences around many Nm sites could be aligned with U3, U8 (SNORD118) or U13 snoRNAs by searching for antisense complementarity with snoRNAs catalogues in the snoDB 25 . Comparison of minimum folding energy (MFE) across snoRNAs and RNA regions encompassing each Nm site (± 7 nt) ( Supplementary Fig. 3) revealed a possible connection between U3 snoRNA and many Nm sites. U3 snoRNA participates in the first stage of rRNA processing 64 and has been reported to exist in multiple molecular complexes, some containing the canonical box C/D components (Fibrillarin, NOP56, NOP58, and SNU13/15.5K). U3 snoRNA complexes have also been reported to shuttle between the nucleus and cytoplasm 65 . Consistent with a model in which noncanonical snoRNAs directly guide a subset of the Nm sites detected in our RibOxi-seq dataset, several of the most prominent Nm peaks overlapped with snoRNA–mRNA chimeras captured by U3 or U8 (SNORD118) in published 293T CLASH data 66 . Four representative examples are shown in Fig. 5 and additional overlaps of CLASH fragments with Nm peaks are shown in Supplementary Fig. 4 . For GUK1, a discrete Nm peak in both H9 and CT2 cells aligns exactly with a U3-derived CLASH fragment, and IntaRNA 67 predicts a stable U3–GUK1 interaction spanning the modified nucleotide with an interaction energy of − 10.5 kcal/mol (Fig. 5 a). A similar pattern is observed at RPL7A, where a strong Nm peak corresponds to a hybrid captured with SNORD118, again supported by a favorable predicted interaction (–9.9 kcal/mol) (Fig. 5 b). Two additional mRNAs, AZIN1 and KMT2A, show the same concordance: each features a sharp Nm peak detected by RibOxi-seq2 that overlaps with U3-captured CLASH reads, and each forms a thermodynamically stable interaction with U3 in IntaRNA modeling (Fig. 5 c,d). Notably, for all four transcripts, the strongest predicted base pairing occurs directly over, or immediately adjacent to the Nm-modified position, although these interactions lack the canonical alignment with a recognizable D-box element. Together (Fig. 5 and Supplementary Data 4 ), these examples are consistent with a model that a subset of mRNAs may acquire 2′-O-methylation through direct interaction with snoRNAs such as U3 and SNORD118, via a noncanonical mechanism. Targets of orphan SNORD113/114 snoRNAs . Since CT2 iNs and CT2-smDEL iNs differ in expression of the orphan SNORD113/114 snoRNA clusters (Fig. 1 b), we asked whether these cells could be used to identify novel targets for these specific snoRNAs. Our approach was to compare Nm profiles, searching for Nm signals in CT2 iNs that are missing in CT2-smDEL iNs as well as in both H9 iNs and H9-smDEL iNs, which also lack expression of the SNORD113/114 region. This was facilitated by the reproducibility of our data. Figure 6 a shows a genomic region witha strong peak in the C12orf57 3’-UTR in CT2 iNs that is completely lacking in the CT2-smDEL data. This site is complementary to SNORD114 with the Nm positioned 5 nucleotides upstream of the box D element, consistent with this site being a canonical target. Nm-VAQ revealed that this site is modified in 24% of the transcripts. Another (Fig. 6 b) in the lncRNA MIAT is seen in 94% of transcripts. A similar strategy allowed us to identify additional SNORD113 and SNORD114 targets including PCDH10, SLC22A17, ANKRD13B, SMG5, PARP6, DDX17 and a number of noncoding transcripts (Fig. 6 c). Importantly, these genes are closely linked to fundamental processes in brain development, and their dysfunction is consistent with mechanistic basis for the onset of neurological disorders such as Kagami-Ogata syndrome. Targets of orphan SNORD116 snoRNAs. Since lack of expression of SNORD116 snoRNAs has been related to Prader-Willi syndrome, the smDEL cell lines with SNORD116 cluster deletions represent a valuable resource to investigate the molecular basis of the disease. In order to qualify as potential canonical sites, there needed to be an Nm signal in both H9 and CT2 iNs but no signal at the same position in both H9-smDEL and CT2-smDEL iNs. Also, the Nm sites needed to be complementary to SNORD116 with the Nm site positioned 5 nucleotides upstream of a box D or D’ element. While we found a number of SNORD113/114 targets, we were only able to identify two potential SNORD116 targets. However, the two targets identified are of considerable interest. These are NLGN3 and DGKK (Fig. 6 d). For each of these mRNAs, there is extensive basepairing with multiple SNORD116 species and the potential Nm sites are 5 nucleotides upstream of the box D element. However, as PWS pathology is closely connected to lack of expression of SNORD116s, our failure to identify numerous robust SNORD116 targets was unexpected. This could be for several reasons. First, some physiological SNORD116 targets may not be expressed well in our iNs. Since PWS is generally thought to be primarily a disorder affecting the hypothalamus, some targets most abundantly expressed there could be poorly expressed in iNs. Second, our findings could indicate that these snoRNAs, which are highly abundant in neurons, may have additional important functions apart from guiding 2’-O-methylation. Third, some PWS pathology may be connected not directly to SNORD116 snoRNAs, but to expression of other noncoding transcripts expressed from this genomic region 68 – 70 . Discussion Here we have used RibOxi-seq2 to generate the first comprehensive picture of the Nm landscape in neurons. Sequencing was carried out on two isogenic pairs of NGN2-induced neuronal cultures derived from embryonic stem cell lines. Each isogenic pair included one wild-type cell line and another harboring a small deletion of 30 related tandem orphan snoRNAs (SNORD116s) from the paternal chr15q11-q13 region, whose loss of expression is associated with Prader-Willi syndrome. The CT2-based isogenic pair also differed in expression of the orphan SNORD113/114 clusters from the imprinted chr14q32.2 region. Thousands of shared Nm sites were identified in these cells, in both coding and noncoding RNAs. The most abundant cellular Nm modifications occur in rRNA. In rRNA, RibOxi-seq2 identified almost all annotated Nm sites but is limited by two technical issues. First, when two Nm sites exist in tandem, RibOxi-seq2 can only see the distal site, except when this site is not fully modified. This led to an inability to monitor several sites in both 18S and 28S rRNA. Second, since RibOxi-seq2 is qualitative and not quantitative, sites modified at a low level even in abundant RNAs such as rRNA can lead to some Nm peaks that are not dramatically higher than background signals. Owing to these issues, we further analyzed ribosomal Nm profiles using RiboMeth-seq, which does not suffer from these drawbacks. Results showed not only that RibOxi-seq2 is highly efficient at identifying rRNA Nm sites, but also that ribosomal RNA in iNs has a distinct modification signature compared to that in embryonic stem cells. In particular, Um354 in neuronal 18S rRNA was seen by both methods and has been reported to be more highly expressed in differentiated cells and directed by SNORD90 55 . This raises the question of how a specific rRNA Nm profile affects translational efficiency or regulation in neurons. Might it affect the translation not only of neuronal mRNAs, but also those harboring neuron-specific Nms? Apart from rRNA, we also observed a number of reported Nm sites in other abundant noncoding RNAs such as snRNAs and tRNAs, showing that RibOxi-seq2 has the potential to monitor such sites in RNA samples. We also identified and validated a novel Nm site in 7SK RNA. Owing to its ability to detect Nm sites even in low-abundance RNAs, RibOxi-seq identified thousands of Nm sites in hundreds of mRNAs and lncRNAs. Importantly, a large fraction of these sites appeared in all four iN cultures, while only a small fraction of Nm sites appeared to be cell- or parent-specific. We don’t yet know the basis of specificity, but we hypothesize that the shared Nm sites include a neuronal signature of genome-wide 2’-O-methylation. While most Nms are shared between the different neuron samples, some are cell-specific. Nm sites in mRNAs are broadly distributed both in functional categories and in regions of internal RNA modification. Thus, about 5% are in 5’-UTRs, 40% in 3’-UTRs, and the rest in coding exons. While some mRNAs may harbor sites that are partially modified, other mRNAs have Nm sites that are almost fully modified. Nm-VAQ revealed that a number of sites in abundant mRNAs are present at almost 100% modification. Such high levels of modification have also been reported recently by others 18 , though most sites identified in this study do not match the positions reported in that work and may thus represent important cell-specific RNA modifications or differences in methodology. What targets most mRNA Nm sites? In our studies, we were surprised to discover that we could not match the great majority of mRNA Nm sites to canonical RNA-snoRNA interactions. This suggested that such modifications may occur via noncanonical interactions, or that many may be directed via recruitment of other activities such as the methyltransferase FTSJ3. Intriguingly, however, we observed that many sites exhibit complementarity to abundant but noncanonical snoRNAs such as U3, U13, and U8 (SNORD118). Do these snoRNAs and their associated proteins harbor methyltransferase activities or have the ability to recruit them? U3 has been reported to exist in multiple distinct snoRNP complexes, and U3 as well as some of its protein components have been shown to shuttle between the nucleus and cytoplasm 65 , 71 , 72 . Our findings with U3 are consistent with the recent report of abundant U3-mRNA interactions using the sno-KARR-seq RNA-RNA interaction method 62 . Importantly, those authors observed that knockdown of U3 led to a reduction in overall levels of mRNA Nm modification. Another group also reported a very high number of U3-mRNA interactions 63 . Together with our CLASH results, these findings lead us to hypothesize that U3, and perhaps also U13 and U8, play an important role in mRNA Nm modification in addition to a role in rRNA maturation. While Nm sites in abundant mRNAs could not be assigned to snoRNA interactions following established rules, our results support the assertion that some Nm peaks do represent canonical snoRNA-mRNA interactions. This possibility is supported by the identification of a number of sites that appear to be canonical targets of previously annotated orphan snoRNAs, including members of the SNORD113, 114, and 116 families. Our data represent the first identification of likely targets of these snoRNAs. Most of these targets are in RNAs that are connected to neurodevelopmental disorders. Deletion of the SNORD113/114 region (chr14q32.2) leads to the rare multi-symptom disorder Kagami-Ogata syndrome 50 , 51 . Specific expression patterns of SNORD113/114 snoRNAs in the brain and strong associations with neurodevelopmental disorders point to crucial and specialized roles in brain development, function, and potentially in the pathogenesis of neurological conditions. SNORD113-6 has been reported to methylate tRNA 73 but our data suggest a possible additional role in mRNA modification. Novel mRNA targets of SNORD113 that we have identified here include PARP6 and DDX17 (Fig. 6 c). PARP6 is a regulator of hippocampal dendritic morphogenesis 74 . DDX17 interacts with and co-regulates the transcriptional repressor element 1-silencing transcription factor (REST), which is a critical regulator of neuronal differentiation, suppressing neuronal gene expression in progenitor cells 75 . SNORD114 targets also shed light on potential roles in neuronal development. C12orf57 has been reported to be a critical gene involved in brain development, particularly the formation of the corpus callosum and the regulation of synaptic function. It is also involved in neurodevelopment and is a regulator of synaptic AMPA currents and excitatory neuronal homeostasis 76 , 77 . Mutations in this gene are associated with developmental brain abnormality 78 . MIATis a lncRNA associated with neuronal development 79 , 80 . PCHD10, a cadherin-related neuronal receptor, is associated with autism and neuropsychiatric disorders 81 , 82 . SLC22A17 (Solute Carrier Family 22 Member 17), also known as the lipocalin-2 receptor, has emerged as a significant player in the brain, particularly in the context of neuroinflammation and neurodegeneration. While initially identified for its role in organic cation transport and iron homeostasis, recent research has highlighted its critical involvement in the blood-brain barrier (BBB) integrity and cell death pathways, especially after stroke 83 . ANKRD13B is a ubiquitin-binding protein that is widely expressed in the brain. Finally, it is noteworthy that SNORD114 appears to target a number of noncoding transcripts, including lncRNAs and miRNA host genes. PKD1P6-NPIPP1 is a cell-cycle related lncRNA and has been implicated as a prognostic marker for hepatocellular carcinoma 84 . Further studies are warranted to investigate the functional ramifications of these Nm modifications. We also found two potential SNORD116 targets, DGKK and NLGN3, and these genes have functions consistent with roles in PWS. NLGN3 (neuroligin 3) is a crucial postsynaptic cell adhesion molecule that plays a pivotal role in organizing and stabilizing synapses and which is linked to autism 85 . It is also a key regulator of gonadotropin-releasing hormone deficiency and is connected to hypogonadism 85 . DGKK (diacylglycerol kinase kappa) converts diacylglycerol to phosphatidic acid, regulating the balance between these two important signaling lipids in the brain. These lipids are involved in neurotransmitter signaling, synaptic plasticity, and overall neuronal communication 86 , 87 . DGKK has been reported to be a (and possibly the) primary target of the Fragile X-related protein FMRP 86 , 88 . Interestingly, both NLGN3 and DGKK are translationally upregulated by FMRP 86 , 89 so the translational consequences of Nm modifications in their mRNAs clearly warrant future study. Thus, both of the potential SNORD116 targets identified here may contribute to the developmental, neurological, and behavioral features of PWS. In fact, among others, both of these genes were hypothesized as possible PWS targets in previous work 90 . In conclusion, we note that the isogenic cell models described here may prove useful in additional studies of Nm modifications and functions. As these cells are embryonic stem cells, they can be differentiated into a multitude of additional cell and organ types, and their differences in orphan snoRNA expression can be exploited to examine the roles of SNORD113, SNORD114, and SNORD116 box C/D snoRNAs in multiple tissue types, both at transcriptomic and targeted Nm levels. Limitations of the study H9 and CT2 cells provide relevant physiological models for investigating PWS and CT2 cells provide a useful model for the study of functions of SNORD113 and SNORD114; however, Nm sites present in low abundance species may be overlooked by the RiboXi-seq2 method. Additionally, as both cell lines are derived from female donors, future studies employing male-derived cell lines will be important. PWS is thought to be largely a disorder connected to the hypothalamus, and SNORD113/114 targets, which may exist in numerous other cell types. By differentiating into different cell and organ types, the CT2/CT2 smDEL isogenic cell pair of human ESCs used here may offer the potential to gain even further insights into the functions of SNORD113, SNORD114, and SNORD116. Whether Nm modifications occur co-transcriptionally or post-transcriptionally; or primarily in the nucleus or in the cytoplasm has not been firmly established. This issue is particularly relevant for U3, which has been reported to be at least partially localized to the cytoplasm. Further studies will be necessary to clarify the mechanisms of U3/U8 (SNORD118)/U13 to mRNA Nm targets. Methods Cell culture Culture and differentiation were carried out as described previously 53 . CT2 and H9 human embryonic stem cells (hESCs) including SNORD116 deletion variants were cultured under feeder-free conditions using Matrigel-coated (Corning, #354277) 100-mm dishes and maintained in mTeSR Plus (STEMCELL Technologies, #100-0276) in a humidified atmosphere at 37°C with 5% CO₂. The culture medium was replaced daily, and cells were passaged approximately every 4–5 days upon reaching 80–90% confluency. For passaging, the medium was removed, and dishes were treated with 0.5 mM EDTA (Invitrogen, #15575020) in PBS, followed by a 2-minute incubation at 37°C. The EDTA solution was then aspirated, and fresh mTeSR Plus medium was added. The cell aggregates were carefully detached using either a StemPro EZPassage Tool (Gibco, #23181010) or by gently scratching the dish with a glass pipette. The passage ratio was maintained at approximately 7–10 to ensure optimal growth. When the hESCs reached 70–80% confluency, cells were prepared for differentiation. First, any differentiated cells were manually removed, and the 100-mm dish was rinsed with PBS. The cells were then treated with Accutase (Millipore, #SCR005) and incubated for 2 minutes at 37°C. After incubation, 10 mL of basal media was added to the cell suspension, which was transferred to a 50-mL conical tube and centrifuged at 1200 rpm for 3 minutes. The supernatant was aspirated, and the pellet was resuspended in 1 mL of Induction Media (IM). The cells were singularized by pipetting up and down 3–5 times using a 1-mL pipette. An additional 9 mL of IM was then added to the suspension. The Induction Medium (IM) was prepared by supplementing DMEM/F12 (with HEPES) (Gibco, #11330032) with 1X N-2 Supplement (Gibco, #17502048), 1X MEM Non-Essential Amino Acids (Gibco, #11140050), and 1X GlutaMAX Supplement (Gibco, #35050061). For differentiation, 4 million cells were plated in a Matrigel-coated (Corning, #354277) 100-mm dish and cultured in IM supplemented with 2 μM Doxycycline Hydrochloride (Fisher Scientific, #BP26535). The cells were fed daily with IM containing 2 μM doxycycline hydrochloride for four days. On day 4 of differentiation, the cells were singularized again using Accutase as described above. The cell pellet was resuspended in Cortical Media (CM), which was prepared by mixing equal volumes of DMEM/F12 with HEPES and Neurobasal Medium (Gibco, #21103049) and supplementing with 1X B27 Supplement (Gibco, #17504044), 10 ng/mL Recombinant Human BDNF Protein (R&D Systems, #248-BD), 10 ng/mL Recombinant Human GDNF Protein (R&D Systems, #212-GD), 10 ng/mL Human NT-3 Recombinant Protein (PeproTech, #450-03), and 1 μg/mL Laminin Mouse Protein (Gibco, #23017015). Cells were plated at a density of 13 million cells per 100-mm dish in CM supplemented with 10 μM ROCK inhibitor Y-27632 dihydrochloride (Tocris, #1254). A complete medium change with CM was performed the following day, and the media was subsequently changed daily until collection on day 11. The 100-mm dishes were pre-coated with 100 μg/mL Poly-D-lysine hydrobromide (PDL, Millipore Sigma, #P0899) and 5 μg/mL laminin. Initially, 6 mL of the 100 μg/mL PDL working solution was applied to each dish and incubated overnight to ensure adequate coating. The next day, the dishes were thoroughly washed twice with PBS to ensure they remained hydrated. Subsequently, 6 mL of the 5 μg/mL laminin working solution was added to the PDL-coated dishes, which were then incubated overnight to prepare them for neuron differentiation. RNA Extraction Total RNA was extracted from cells using the TRIzol reagent protocol. Briefly, cells were washed with PBS, pelleted by centrifugation, and lysed in TRIzol reagent (Invitrogen, #15596026). After incubation at room temperature for 5 minutes, 0.2 mL of chloroform (Invitrogen, #288306) per mL of TRIzol was added, followed by vigorous shaking and centrifugation at 12,000 × g for 15 minutes at 4°C. The aqueous phase containing RNA was collected, mixed with 0.5 mL of isopropanol (Invitrogen, # I9030) per mL of TRIzol, incubated for 10 minutes, and centrifuged at 12,000 × g for 10 minutes at 4°C. The resulting RNA pellet was washed with 75% ethanol, air-dried, and resuspended in RNase-free water. RNA cleanup and DNase I treatment were performed using the RNA Clean & Concentrator-25 (Zymo Research, #R1017) according to the manufacturer’s instructions. RNA Ethanol Precipitation To 100 µL of RNA solution, 1 µL of Linear Acrylamide (250–500X; Invitrogen, AM9520) and 250 µL of ice-cold 100% ethanol (2.5–3.0 volumes) were added. The mixture was thoroughly mixed and stored at −80°C for 1 hour or overnight at −20°C to allow RNA precipitation. The precipitated RNA was recovered by centrifugation at 14,000 × g for 20 minutes at 4°C. The supernatant was removed, and the RNA pellet was washed with 0.5 mL of ice-cold 75% ethanol, followed by centrifugation at 10,000 × g for 5 minutes at 4°C. Residual ethanol was carefully removed using a 20-µL pipette after a brief spin-down, and the pellet was air-dried on ice for 5–10 minutes before being resuspended in nuclease-free water. DNA Ethanol Precipitation To 100 µL of DNA solution, 10 µL of 3M Sodium Acetate (Invitrogen, #AM9740), 1 µL of GlycoBlue Coprecipitant (Invitrogen, #AM9515), and 250 µL of ice-cold 100% ethanol (2.5–3.0 volumes) were added. The solution was thoroughly mixed and stored overnight at −20°C to allow DNA precipitation. The DNA was recovered by centrifugation at 14,000 × g for 20 minutes at 4°C. The supernatant was carefully removed, and the DNA pellet was washed with 0.5 mL of ice-cold 75% ethanol, followed by centrifugation at 10,000 × g for 5 minutes at 4°C. Residual ethanol was carefully removed using a 20-µL pipette after a brief spin-down, and the pellet was air-dried on ice for 5–10 minutes before being resuspended in nuclease-free water. mRNA Isolation mRNA was isolated from 1-1.5 mg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, #E7490L) following the manufacturer's protocol. After the isolation procedure, the purified mRNA was collected by transferring 88 μL of the supernatant to a 1.5 mL DNA LoBind Tube (Eppendorf, #0030108051). The integrity of the isolated mRNA was assessed using RNA ScreenTape and RNA ScreenTape Sample Buffer (Agilent, #5067-5576 and #5067-5577). RibOxi-seq2 Methods These were essentially as described 39 but with a number of modifications. RNA Fragmentation A 0.25 U/μL benzonase working solution was prepared by mixing 100 μL of 10× benzonase buffer, 899 μL of nuclease-free water, and 1 μL of 250 U/μL Benzonase Nuclease (Millipore Sigma, #E8263). For fragmentation, 88 μL of 10–20 μg mRNA was heated at 95°C for 3 minutes in a ThermoMixer C (Eppendorf) and immediately placed on ice for 3 minutes. Subsequently, 10 μL of 10× benzonase buffer and 2 μL of the benzonase working solution were added to the mRNA solution. The mixture was vortexed, briefly centrifuged, and immediately incubated on ice for 80 minutes. The digestion time requires optimization, particularly for different cell lines and different batches of benzonase. Post-fragmentation: 100 μL of nuclease-free water and 200 μL of Acid-Phenol:Chloroform (pH 4.5; Invitrogen, #AM9722) were added to the sample. The mixture was vortexed for 15–20 seconds, incubated at room temperature for 2 minutes, and centrifuged at 18,000 × g for 10 minutes at 4°C. The aqueous phase (180–190 μL) was carefully transferred to a new tube. RNA ethanol precipitation was performed as described above. The final RNA was suspended in 64 μL of nuclease-free water. RNA quality was assessed using 1 μL of the sample, confirming the presence of a sharp peak between 25–200 nucleotides, skewing to the right. RNA Oxidation, β-Elimination, and Dephosphorylation A 200 mM Sodium meta-periodate (NaIO4; Thermo Fisher, #20504) solution was prepared by dissolving 42.78 mg of NaIO4 in 1 mL of nuclease-free water. The solution was protected from light, kept on ice, and used the same day. For the oxidation reaction, 64 μL of fragmented RNA was mixed with 8 μL of oxidation-elimination buffer (pH 8.5) and 8 μL of NaIO4 (200 mM), resulting in a final volume of 80 μL. The mixture was vortexed, briefly centrifuged, and incubated at 37°C with shaking (350 rpm) for 45 minutes in a ThermoMixer C (Eppendorf). Following oxidation, the RNA was purified using ethanol precipitation, and cleaned up using Micro Bio-Spin P-6 Gel Column (Bio-Rad, #7326221), with the RNA eluted in 84 μL of nuclease-free water. The oxidation-elimination buffer was prepared from 2M L-Lysine monohydrochloride (Sigma-Aldrich, #L5626) in nuclease-free water. The pH was adjusted to 8.5 using 2 M NaOH, and the solution was filtered through a 0.22 μm filter. For the dephosphorylation reaction, 84 μL of oxidized RNA was mixed with 10 μL of rCutSmart buffer (10×, New England Biolabs, #B6004S), 2 μL of RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019), and 4 μL of Quick CIP (5 U/μL, New England Biolabs, #M0525S), resulting in a final volume of 100 μL. The reaction was incubated at 37°C for 10 minutes, followed by heat inactivation at 80°C for 2 minutes. The RNA was subsequently purified using ethanol precipitation. The entire oxidation, β-elimination, and dephosphorylation process was repeated three additional times. Subsequently, RNA cleanup was performed using the Zymo RNA Clean & Concentrator-25 (Zymo Research, #R1017). The final RNA was eluted in 66 μL of nuclease-free water. Further dephosphorylation reactions involved two stages. In stage I, 66 μL of oxidized RNA was mixed with 8 μL of T4 PNK buffer (10×, pH 6.0), 4 μL of T4 polynucleotide kinase (10 U/μL, New England Biolabs, #M0201), and 2 μL of RNaseOUT inhibitor, resulting in a total volume of 80 μL. The mixture was incubated at 37°C for 3 hours. In stage II, 80 μL of oxidized RNA from the first reaction was combined with 10 μL of T4 PNK buffer (10×, pH 7.6), 4 μL of T4 Polynucleotide Kinase (10 U/μL, NEB, #M0201S), 20 μL of 10 mM Adenosine 5'-Triphosphate (ATP; New England Biolabs, #P0756S), and 66 μL of nuclease-free water to a final volume of 180 μL. This reaction was incubated at 37°C for 1 hour and inactivated with the addition of 2 μL of 0.5 M EDTA. The RNA was purified by ethanol precipitation and resuspended in 70 μL of oxidation-only buffer. The Oxidation-only buffer was prepared with 4.375mM Sodium Tetraborate Decahydrate (Fisher Scientific, #BP175-500), 50mM Boric Acid (Fisher Scientific, #A74-1), and nuclease-free water. The pH was adjusted to 8.6 using 2 M NaOH, and the solution was filtered through a 0.22 µm filter before use. An additional oxidation reaction was performed by combining 70 μL of oxidized RNA with 10 μL of NaIO4 solution (200 mM) to a final volume of 80 μL. The mixture was incubated at 37°C with shaking (350 rpm) for 45 minutes. The oxidized RNA was purified using ethanol precipitation. Final cleanup steps included the use of Micro Bio-Spin P-6 Gel Columns and the Zymo RNA Clean & Concentrator-5 (Zymo Research, #R1013), with the RNA eluted in 20 μL of nuclease-free water. 3’ -DNA Linker Ligation The 3’-DNA linker ligation was performed using 20 μL of oxidized RNA, 2 μL of a 3’-DNA linker (10 μM), 2 μL of RNaseOUT inhibitor, 17 μL of 50% PEG 8000, 5 μL of RNA ligase buffer (10×, New England Biolabs, #B0216S), and 4 μL of T4 RNA Ligase 2 truncated KQ (New England Biolabs, #M0373), resulting in a total reaction volume of 50 μL. The reaction mixture was thoroughly mixed, briefly centrifuged, and incubated overnight at 16°C for 18 hours in a thermal cycler. The RNA was subsequently purified using ethanol precipitation and resuspended in 15 μL of nuclease-free water. TBE-Urea Gel Preparation Gel purification was performed to separate the ligation products from free 3’ DNA linkers. Two stock solutions were prepared for the TBE-Urea gel. Solution A was made by dissolving 500 g of Urea (Bio-Rad, #1610745) in 500 mL of 40% Acrylamide Solution (Bio-Rad, #1610140) and 100 mL of 10× TBE buffer (Bio-Rad, #1610770). The volume was brought up to 1,000 mL using 1× TBE buffer. Solution B was prepared similarly by dissolving 500 g of Urea in 100 mL of 10× TBE buffer and 500 mL of nuclease-free water, with the final volume adjusted to 1,000 mL using 1× TBE buffer. Both solutions were filtered through a 0.22 μm filter. A 15% TBE-Urea gel was prepared by mixing 37.5 mL of Solution A, 12.5 mL of Solution B, 400 μL of 10% ammonium persulfate (APS; Bio-Rad, #2610700), and 37.5 μL of TEMED (Bio-Rad, #161-0800) to a final volume of 50.4 mL. The mixture was immediately poured between the glass plates of the Bio-Rad Mini-Protean gel casting apparatus, and a comb was inserted. The gel was allowed to polymerize at room temperature for 60 minutes. Prior to electrophoresis, 2× RNA loading dye (New England Biolabs, #B0363S) was added to the RNA sample, which was then incubated at 72°C for 2 minutes and placed on ice for 3 minutes. The samples were loaded, and electrophoresis was performed at 20W for 50 minutes. The gel was stained with SYBR Gold Nucleic Acid Gel Stain (Invitrogen, #S11494) by incubating it in a 1× TBE staining solution for 15 minutes with gentle shaking. RNA bands were visualized using a Dark Reader Non-UV Transilluminator (Model: DR-46B), and the desired fragment was excised and transferred to a tube. Images of the gel were captured both before and after excision. Acrylamide Gel RNA Extraction To extract RNA product from the acrylamide gel, LoBind tubes were prepared with 1.5 μL GlycoBlue Coprecipitant and 33 μL Sodium Acetate. A hole was pierced in the bottom of a 0.6 mL tube using an 18G needle, holding the needle near the tip and carefully pressing it into the tube’s bottom. The 0.6 mL tube was placed inside a 2 mL tube, and the gel slices were added to the 0.6 mL tube. The assembly was centrifuged at maximum speed for 1 minute at 4°C, allowing the crushed gel to flow into the larger tube. Each tube was supplemented with 300 μL of nuclease-free water, and the mixture was incubated in a ThermoMixer C (Eppendorf) at 70°C for 10 minutes with shaking at 1800 rpm. This incubation was repeated an additional two times. Following incubation, the tubes were briefly centrifuged, and the supernatant was transferred to a Costar SpinX Centrifuge Tube Filter (0.45 μm; #8162). The SpinX tube was centrifuged at 10,000 rpm for 1 minute, and the flow-through was transferred into prepared LoBind tubes containing GlycoBlue and sodium acetate. Ethanol precipitation was performed, and the RNA product was eluted in 22 μL of nuclease-free water. 5’ -RNA Linker Ligation To perform the 5’-RNA linker ligation, 2 µL of 5’-RNA linker (50 µM) was denatured at 72°C for 2 minutes and immediately placed on ice. A ligation reaction mixture was prepared by combining 22 µL of the ligated RNA product, 4 µL of 100% DMSO, 4 µL of T4 RNA Ligase Reaction Buffer (10×, B0216S), 4 µL of ATP (10 mM), and 1 µL of RNaseOUT inhibitor, resulting in a total volume of 35 µL. Next, 35 µL of the prepared ligation mixture was combined with 3 µL of T4 RNA Ligase 1 (New England Biolabs, #M0204S) and 2 µL of the 5’-RNA linker (50 µM), bringing the final reaction volume to 40 µL. The reaction was mixed, briefly centrifuged, and incubated at 25°C for 1 hour. Following the incubation, RNA purification was carried out using the Monarch Spin RNA Cleanup kit (New England Biolabs, #T2030) according to the manufacturer’s instructions, and the RNA product was eluted in 20 µL of nuclease-free water. cDNA Synthesis For cDNA synthesis, 20 µL of the ligated RNA product was combined with 1 µL of 10 µM Reverse Transcriptase (RT) primer, 2 µL of 10 mM dNTPs, and 2 µL of nuclease-free water in a final volume of 25 µL. The mixture was denatured at 65°C for 5 minutes, immediately placed on ice, and briefly centrifuged. To this, 8 µL of ProtoScript II buffer (5×, New England Biolabs, #B0368S), 1 µL of RNaseOUT inhibitor, 4 µL of 0.1 M DTT (New England Biolabs, #B1034A), and 2 µL of ProtoScript II Reverse Transcriptase (New England Biolabs, #M0368S) were added to bring the final reaction volume to 40 µL. The reaction was incubated at 50°C for 1 hour to synthesize the first strand of cDNA. To hydrolyze the RNA, 4 µL of 1 N Sodium Hydroxide Solution (Fisher Chemical, #SS266-1) was added, and the mixture was incubated at 95°C for 15 minutes. The reaction was neutralized with 4 µL of 1 M Tris–HCl (pH 7.5; Invitrogen, #15567027), and the cDNA was purified using 1.8× AMPure XP Reagent (Beckman Coulter, #A63881) following the manufacturer’s protocol. cDNA Purification Using AMPure XP Reagent The cDNA was purified by adding 1.8× AMPure XP reagent to the cDNA sample (48 µL cDNA + 86.4 µL beads). The mixture was thoroughly mixed by pipetting up and down 10 times and incubated at room temperature for 10 minutes. The tubes were briefly spun down and placed on a magnetic stand for 5 minutes until the liquid became clear. The supernatant was carefully removed and discarded, and the DNA-bound beads were washed twice with 500 µL of freshly prepared 80% ethanol. After each wash, the beads were incubated on the magnetic stand for 30 seconds, and the supernatant was discarded. Residual ethanol was carefully removed with a pipette, and the beads were air-dried for 5–10 minutes. The dried beads were resuspended in 32 µL of nuclease-free water, incubated at room temperature for 2 minutes, and briefly spun down. The eluate (30 µL), containing the purified cDNA, was carefully transferred to a new tube for subsequent use. Library Amplification and Library Quality Control (QC) Library amplification was performed using dual-index sequences. The reaction was set up with 30 µL of cDNA, 10 µL of 5× Q5 Reaction Buffer, 1 µL of 10 mM dNTPs, 2.5 µL of 10 µM i7 index, 2.5 µL of 10 µM i5 index, 0.5 µL of Q5 High-Fidelity DNA Polymerase (New England Biolabs, #M0491), and 3.5 µL of nuclease-free water, in a final volume of 50 µL. Thermocycling conditions were as follows: initial denaturation at 98°C for 30 seconds, followed by three cycles of 98°C for 10 seconds, 52°C for 30 seconds, and 72°C for 30 seconds. This was followed by 18 cycles of 98°C for 10 seconds, 62°C for 30 seconds, and 72°C for 30 seconds. The final extension was performed at 72°C for 2 minutes, and the reaction was held at 4°C. The amplified library was purified using ethanol precipitation and P-6 columns, followed by size selection using SPRIselect beads (Beckman Coulter, #B23318) to ensure high-quality libraries. SPRI-Based Size Selection SPRI-based size selection was performed using SPRIselect (Beckman Coulter, #B23318) to ensure appropriate library fragment size distribution. For left-side selection, 60 µL of SPRIselect beads (1.2× ratio) was added to a 50 µL amplified library sample. The beads were thoroughly mixed by pipetting up and down 10 times and incubated at room temperature for 1 minute. The reaction tube was then placed on a magnetic stand for 5–10 minutes to allow the beads to settle. Once the liquid clarified, the supernatant was carefully discarded. The beads were washed twice with 500 µL of freshly prepared 85% ethanol. After each wash, the supernatant was removed following a 30-second incubation, and the beads were air-dried for 5–10 minutes to ensure complete ethanol removal. The purified DNA was eluted by resuspending the beads in 52 µL of nuclease-free water. After incubation for 1 minute and magnetic separation, the eluate (50 µL) was carefully collected, leaving the beads behind. For right-side size selection (0.7×/1.1×), 35 µL of SPRIselect beads (0.7× ratio) was added to the 50 µL library sample and mixed thoroughly. Following incubation and magnetic separation, the supernatant containing the desired DNA fragment was transferred to a new reaction tube, and the discarded beads contained larger fragments. To the supernatant, 55 µL of SPRIselect beads (1.1× ratio) was added to remove smaller fragments. After incubation, magnetic separation, and two ethanol washes, the beads were air-dried, and the DNA was eluted in 22 µL of Resuspension Buffer (Illumina, #15026770). The final eluate containing the size-selected library was carefully transferred to a new LoBind tube and stored at −80°C for subsequent analysis. RibOxi-Seq2 Library Preparation and Sequencing Library size distribution was confirmed using the D1000 ScreenTape and reagents on the 4200 TapeStation system (Agilent, #5067-5582 and #5067-5583). Library concentration was quantified using the Qubit dsDNA BR Assay Kit (Invitrogen, #Q32853) on a Qubit 2.0 fluorometer (Invitrogen). For sequencing, libraries were initially run on an Illumina MiSeq system using the MiSeq Reagent Nano Kit v2 (300 cycles; Illumina, #MS-103-1001) with a loading concentration of 9 pM. For higher throughput sequencing, libraries were diluted to a final concentration of 1050 pM and sequenced on an Illumina NextSeq 2000 system using the NextSeq 2000 P2 Kit (200 cycles; Illumina, #20046812). A sequencing depth of 30 million reads per sample was achieved to ensure sufficient coverage for downstream analysis. RibOxi-Seq2 analysis RibOxi-Seq data were processed following the previously established pipeline (https://github.com/yz201906/RibOxi-seq, https://github.com/xxy103/RibOxi-seq) (2). In brief, sequencing adapters were trimmed using Cutadapt v2.7 (https://github.com/marcelm/cutadapt). Paired-end reads were subsequently merged into single-end reads with PEAR v0.9.11. Unique molecular identifiers (UMIs), consisting of 10 randomized nucleotides, were extracted and appended to the fastq headers using the “move_umi.py” script. Quality-filtered reads (with length longer than 20nt, Q-value over 20) were then mapped to the reference genome GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta using STAR v2.7.1a. The aligned reads were filtered and de-duplicated using UMI-TOOLS v1.1.1. Single-nucleotide 3’-end coverage profiles indicative of Nm sites were calculated using Samtools v1.9 and Bedtools v2.29.0. rRNA, snRNA, and tRNA rRNA and snRNA sequences were retrieved from https://rnacentral.org/, tRNA sequences were downloaded from https://gtrnadb.org/. Snoscan RNA modification prediction The annotated snoRNA sequences were downloaded from Human snoDB 25 , the top190 expressed human snoRNAs in 5 human cell lines (HepG2, HEK293T, PC3, A549, MDA-MB-231) were obtained from previous publication 62 . Putative RNA modification sites within these snoRNAs were predicted with snoScan v1.0 61 , using as reference the Nm regions (± 7nt) identified through RibOxi-Seq2 analysis. SnoRNA-RNA interactions The snoRNA-RNA base pairing was computed with snoDB online tools (https://bioinfo-scottgroup.med.usherbrooke.ca/snoDB/sequence_similarity_search/) 25 , predicted interactions were filtered based on alignment scores and thermodynamic plausibility. The RNA-RNA interactions Minimum Folding Energy (MFE) was determined using RNAduplex (ViennaRNA v2.5.1) 91 , which computes the MFE of hybridization between snoRNA and Nm regions (± 7nt) identified from RibOxi-Seq2 analysis. Metagene analysis Metagene plot summarizing the mRNA features were generated with metaPlotR. (https://github.com/olarerin/metaPlotR) RiboMeth-Seq H9 hESCs were maintained on Matrigel-coated (Corning, #354277) plates with Essential 8 media (Thermo Fisher, #A1517001). Stem cells were differentiated to neurons for 14 days per the i 3 neuron protocol 92 . hESCs were prepared for differentiation once 70-80% confluent. Differentiated cells were manually removed. Stem cells were released from the plate with Accutase (Sigma-Aldrich, #SCR005) incubation. Media was added to the cell solution and cells were centrifuged. Cells were resuspended in induction media (IM) supplemented with 2µg/ml doxycycline (Dox) (Milipore Sigma, #D9891) and 10µM Rock Inhibitor (Tocris, #1254). IM was made with DMEM/F12 with HEPES (Thermo Fisher, #11330032), N2 supplement (Thermo Fisher, #17502048), MEM Non-essential amino acids (Thermo Fisher, #11140050), and L-Glutamine (Thermo Fisher, #25030081). Cells were plated at 1.5x10 5 cells/well in a 6-well plate. Cells were fed daily with IM and received 2 additional days of Dox treatment. Cells were then replated in IM on poly-D-lysine/laminin-coated 6-well plates (Millipore Sigma P1149, Fisher Scientific, #23-017-015) at 5x10 5 cells/well. The day after plating, the media was fully changed to cortical neuron culture media (CM) which is composed of DMEM/F12 with HEPES and Neurobasal medium (Thermo Fisher, #21103049) supplemented with laminin, BDNF (Thermo Fisher, #450-02-10UG), GDNF (Thermo Fisher, #450-10-10UG), NT3 (Thermo Fisher, #450-03-10) and B27 supplement (Thermo Fisher, #17504044). Subsequently, half media changes with CM were performed on every other day until the 14 th day of differentiation. Cells were rinsed with PBS and then were collected in TRIzol Reagent (Thermo Fisher, # 15596018). 0.2ml of chloroform per 1ml of TRIzol was added and samples were centrifuged. The aqueous phase was transferred to a new tube and 0.5ml of isopropanol was added. Samples were incubated at -80°C for 10 minutes and then centrifuged to pellet the RNA. The pellet was washed with 75% ethanol and resuspended in nuclease-free water. Samples were prepared in triplicate for RiboMeth-Seq following 37 with some modifications. Briefly, 250ng RNA per sample was subjected to alkaline hydrolysis. Fragmented RNA was then ethanol precipitated and run on a TapeStation to check size distribution. Samples were 3’ end dephosphorylated by incubating for 3 hrs at 37°C with T4 PNK (New England Biolabs, M0201S) and pH6 T4 PNK buffer. Then 5’ end phosphorylation was achieved by adding additional T4 PNK, T4 PNK reaction buffer, and ATP and incubating for 30 minutes. A 3’-DNA linker was ligated to the RNA with T4 RNA ligase 2 truncated KQ (NEB, M0373S). Then a 5’-RNA linker was ligated to the RNA. The linker was denatured and then the ligation reaction was prepared utilizing T4 RNA ligase 1 (NEB, M0204S. cDNA was synthesized using Superscript III (Thermo Fisher, #18080044). The RNA was hydrolyzed and the cDNA purified with AmpureXP beads (Beckman Coulter, A63880). Libraries were amplified with KAPA HiFi DNA polymerase (Roche, #07958927001) and sequenced on Illumina NovaSeq. Adaptor sequences, reverse transcription primers, and PCR primers were applied according to the procedures described above. RiboMeth-Seq Analysis by MethScore was calculated as described 35,93 . The 2 nucleotides flanking each position were used as the flanking region. Briefly, across all positions, the sum of 3’-ends’of profile at the n position and the 5’-ends of profile at the n+1 position was calculated. For each position, the sum of ends was divided by ½ of the sum of the weighted end sum for the flanking positions divided by the sum of the weights. This was subtracted from 1 to give the MethScore. Standard deviation was calculated across 3 replicates. Nm-VAQ Quantitation 500 ng of total RNA from CT2-NGN2 neuron cells was mixed with 50 pmol of the RNA/DNA chimera. The volume was adjusted to 11 µl using 10 µM Tris pH 7.0 buffer. For mRNA-specific targeting, 500 ng of polyA-selected RNA (NEB, #E7490L) was used. Samples were incubated at 95°C for 1 minute and immediately transferred to ice. Subsequently, 5 µl of the annealed RNA/chimera mixture was combined with 1 µl of RNase H enzyme (NEB, #M0297S), 1 µl of 10x RNase H buffer (NEB, #B0297S), and 3 µl of nuclease-free water. The remaining 5 µl of the annealed RNA/chimera mixture was mixed with 1 µl of 10x RNase H buffer and 4 µl of nuclease-free water. The samples were thoroughly mixed by pipetting and incubated at 37°C for 30 minutes. At this stage, the protocol diverges for highly abundant rRNA and low abundance mRNA. For highly abundant RNAs, the samples were incubated at 90°C for 10 minutes to denature the RNase H enzyme, and then placed on ice. After denaturation, the samples were diluted 1:5. Then, 1 µl of the diluted sample was used for cDNA synthesis with SuperScript III Reverse Transcriptase (Invitrogen, #18080044) and random hexamers. Subsequently, 1 µl of cDNA was used for RT-qPCR with PowerSybr (Applied Biosystems, #4368706). For low abundance mRNA, the reaction volume after the 30-minute RNase H cleavage step was increased to 30 µl with sterile nuclease-free water, and 30 µl of Phenol-chloroform-isoamyl alcohol mixture (Millipore Sigma, #77617) was added. After vigorous vortexing, the mixture was centrifuged at 12,000 g for 5 minutes. Approximately 20 µl of the upper aqueous phase was transferred to a clean 1.7 mL Eppendorf tube. Cytiva Microspin G-50 columns (Cytiva, #27533001) were prepared by loosening the cap, removing the bottom plug, and placing them into 2 mL collection tubes. Excess buffer was removed by centrifuging at 700 g for 1 minute. The column was then transferred to a clean 1.7 mL Eppendorf tube, and the upper phase from the previous extraction was added to the column. To elute, the tube was centrifuged at 700 g for 2 minutes. 1.5 µl of the eluate was used for cDNA synthesis with SuperScript III and random hexamers. Finally, 1 µl of the cDNA was used for RT-qPCR with PowerSybr. Declarations Correspondence Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Gordon G. Carmichael ( [email protected] ). Code availability RibOxi-Seq2 data for iNs have been deposited at GEO with Bioproject accession number PRJNA1348454. RibOxi-seq data for 293T cells have been deposited at GEO with accession number GSE188194. The scripts for our RibOxi-Seq2 pipeline are available from https://github.com/yz201906/RibOxi-seq. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Acknowledgements This work was supported by NIH grants R35GM118140 to B.R.G., HD099975 and an award from the Foundation for Prader Willi Research to G.G.C., R35GM146883 to J.D.B, F31HD114435 to S.A.A. and R01GM135383 to C.L.H. We thank Sara Olson for helpful suggestions regarding the Illumina sequencing. 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Front Genet 11:38. https://doi.org:10.3389/fgene.2020.00038 Additional Declarations There is NO Competing Interest. Supplementary Files FileS1.xlsx Supplementary Data 1. Nm sites in 18S and 28S rRNA identified by RibOxi-Seq2 and RiboMeth-Seq, related to Fig. 2. FileS2.xlsx Supplementary Data 2. Nm sites in CT2 and H9 cell lines identified by RibOxi-Seq2, related to Fig. 3. FileS3.xlsx Supplementary Data 3. Nm-VAQ quantifications, related to Fig. 4, Fig. 6, Supplementary Fig. 2 and Methods. FileS4.pdf Supplementary Data 4. Nm-VAQ oligos and qPCR-primers, related to Fig. 4, Fig. 6, Supplementary Fig. 2 and Methods. Suppl.figures.pdf Supplementary Figures 1,2 3 and 4 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8523796","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":575564810,"identity":"ce58ce0e-e97c-4b63-9e83-d1b1df022153","order_by":0,"name":"Gordon Carmichael","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYLCCBIYDDAzszAcYGBsgXCK1MLMlkKCFAayFx4A4LfztZ59ueFBzR56fmeebxM8ddgz87DkGeLVInEk3u5Fw7JnhzGbebZK9Z5IZJHve4NdiwJDGdiOx4TDjhsO826QZ2w4wGNwgYIsB/zOwFvv9h3megbXYE9QiAbElcQMzDxvEFglCfrkBtCXh2OHkGYfZjC1725J5JM48K8Crhb8/je3mj5rDtv3tzQ9v/Gyzk+NvT96AVwsG4CFN+SgYBaNgFIwCrAAAu+BIX7U0JtMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0379-6580","institution":"University of Connecticut Health Center","correspondingAuthor":true,"prefix":"","firstName":"Gordon","middleName":"","lastName":"Carmichael","suffix":""},{"id":575564811,"identity":"9aed64f2-d7b3-42be-938c-5a117f427140","order_by":1,"name":"Xuan Ye","email":"","orcid":"","institution":"University of Connecticut Health Center","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Ye","suffix":""},{"id":575564812,"identity":"00d84cb0-4074-4076-9c90-ad39c4bb5df9","order_by":2,"name":"Yaling Liu","email":"","orcid":"","institution":"University of Connecticut Health Center","correspondingAuthor":false,"prefix":"","firstName":"Yaling","middleName":"","lastName":"Liu","suffix":""},{"id":575564813,"identity":"29300430-34d4-4d02-8a44-bb00b07d71e8","order_by":3,"name":"Yinzhou Zhu","email":"","orcid":"https://orcid.org/0000-0003-0043-1321","institution":"Duke University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yinzhou","middleName":"","lastName":"Zhu","suffix":""},{"id":575564814,"identity":"1f342cd7-8979-45a6-9f05-7f6b5e38fadc","order_by":4,"name":"Saran Alshawi","email":"","orcid":"","institution":"University of Connecticut Health Center","correspondingAuthor":false,"prefix":"","firstName":"Saran","middleName":"","lastName":"Alshawi","suffix":""},{"id":575564815,"identity":"fedb7e96-f18c-4208-9698-4de07199dcab","order_by":5,"name":"Brittany Elliott","email":"","orcid":"https://orcid.org/0000-0001-5907-1231","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"Brittany","middleName":"","lastName":"Elliott","suffix":""},{"id":575564816,"identity":"f5252d87-b56a-41d5-b86a-4f9940fda524","order_by":6,"name":"Christopher Holley","email":"","orcid":"https://orcid.org/0000-0002-2870-3352","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Holley","suffix":""},{"id":575564817,"identity":"6238f97c-d830-43f3-8708-294972b4c57a","order_by":7,"name":"Jean-Denis Beaudoin","email":"","orcid":"","institution":"University of Connecticut Health Center","correspondingAuthor":false,"prefix":"","firstName":"Jean-Denis","middleName":"","lastName":"Beaudoin","suffix":""},{"id":575564818,"identity":"f83342e9-2d86-43e8-b81e-d99350fe8ca1","order_by":8,"name":"Brenton R. 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08:43:41","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":236947,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/88f3a073f2c1b4175455d66f.html"},{"id":100594986,"identity":"c417a9c1-83b8-4576-a279-796f07395445","added_by":"auto","created_at":"2026-01-19 13:46:52","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eThe RibOxi-seq2 method to map Nm sites. See Methods for details. RNA species with or without a 3′-end Nm modification were subjected to periodate oxidation. RNAs containing a 3′-end Nm modification supported linker ligation, whereas RNAs lacking the modification did not. \u003cstrong\u003eB. \u003c/strong\u003eUCSC genome browser image of chromosome 15 illustrating representative bigWig tracks from CT2 and H9 induced neurons and from small-deletion induced neurons. Dashed box outlines the deleted region. \u003cstrong\u003eC\u003c/strong\u003e. UCSC genome browser image of chromosome 14q32 illustrating representative bigWig tracks from CT2 and H9 induced neurons and from small-deletion induced neurons. Red tracks represent RNA signal on the sense strand, while blue tracks represent RNA signal on the antisense strand.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/5285ddd1e069ef9e1db2e8d9.jpeg"},{"id":100560565,"identity":"3cdc2da9-f65c-47ee-916b-daff66f445b7","added_by":"auto","created_at":"2026-01-19 08:43:48","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":949044,"visible":true,"origin":"","legend":"\u003cp\u003eNm profiling of rRNA. \u003cstrong\u003eA\u003c/strong\u003e. RibOxi-seq2 peaks in 18S rRNA from induced neurons. Every peak shown aligns with a validated or annotated site. \u003cstrong\u003eB\u003c/strong\u003e. RibOxi-seq22 peaks in 28S rRNA from induced neurons. Every peak shown aligns with a validated or annotated site. \u003cstrong\u003eC\u003c/strong\u003e. RiboMeth-seq results for 18S rRNA from H9 HESCs. \u003cstrong\u003eD\u003c/strong\u003e. RiboMeth-seq results for 28S rRNA from H9 HESCs. \u003cstrong\u003eE\u003c/strong\u003e. RiboMeth-seq results for 18S rRNA from H9 induced neurons s. Note U354m. \u003cstrong\u003eF\u003c/strong\u003e. RiboMeth-seq results for 28S rRNA from H9 induced neurons. Positions are numbered; nucleotide identity is indicated. \u003cstrong\u003eG\u003c/strong\u003e. Nm sites identified in tRNAs. Supporting data is in Figure S2. \u003cstrong\u003eH.\u003c/strong\u003e Nm sites detected by RibOxi-seq2 in snRNAs and 7SK RNA are highlighted in green, while reference Nm sites are indicated in both black and green. Supporting data is in Figure S2.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/184fd61ed479bd209af0a5d7.jpeg"},{"id":100560203,"identity":"9b298a9a-9439-4bbc-ad75-67a1b5c5a6b0","added_by":"auto","created_at":"2026-01-19 08:43:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1122295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. An example of the reproducibility of RibOxi-seq2. UCSC Genome Browser image of a 2 Mb region on chromosome 12 showing representative bigWig tracks from CT2 and H9 induced neurons and from small-deletion induced neurons. RibOxi-seq2 peaks indicate Nm positions, and gene annotations are displayed below. \u003cstrong\u003eB\u003c/strong\u003e. Venn diagram illustrating overlapping Nm sites across induced neuron samples; based on the top 500 Nm peaks. \u003cstrong\u003eC\u003c/strong\u003e. Metagene profile of Nm sites in human mRNA, based on the top 500 Nm sites. \u003cstrong\u003eD, E\u003c/strong\u003e. Representative example of two genes with differing Nm positions between H9 and CT2 induced neurons.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/3a27549e67ae9de4fd30a969.jpeg"},{"id":100595557,"identity":"560fc221-bcf5-4bb5-b1be-79755d55db69","added_by":"auto","created_at":"2026-01-19 13:48:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":624220,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative examples of genes displaying differing Nm positions in CT2 and in small-deletion induced neurons. Percentages adjacent to the peaks denote modification levels quantified by Nm-VAQ. Red tracks represent RNA signal on the sense strand, while blue tracks represent RNA signal on the antisense strand. \u003cstrong\u003eA\u003c/strong\u003e. \u003cem\u003eACTB\u003c/em\u003e; Nm peaks in exons 4 and 5. \u003cstrong\u003eB\u003c/strong\u003e. \u003cem\u003eSNCG\u003c/em\u003e; Nm peak in exon 3. \u003cstrong\u003eC\u003c/strong\u003e. \u003cem\u003eFAM171A1\u003c/em\u003e; Nm peak in 3’-UTR. \u003cstrong\u003eD\u003c/strong\u003e. Nm peak in \u003cem\u003eMT-CO1\u003c/em\u003e. \u003cstrong\u003eE\u003c/strong\u003e. \u003cem\u003eTPT1\u003c/em\u003e; Nm peak in 5’-UTR. \u003cstrong\u003eF\u003c/strong\u003e. Quantitation of Nm peaks using Nm-VAQ. Additional data are in Table S3.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/ee740e8f89894b1528dab8e7.jpeg"},{"id":100560509,"identity":"6ca449b0-fdaf-494a-a666-e94e123848a7","added_by":"auto","created_at":"2026-01-19 08:43:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":772022,"visible":true,"origin":"","legend":"\u003cp\u003eRibOxi-seq2 Nm mapping and snoRNA-guided IntaRNA predictions for sites captured in snoRNA-specific CLASH. (\u003cstrong\u003eA, C, E, G\u003c/strong\u003e) RibOxi-seq2 tracks show 3′ Nm read counts across four neuron cell line conditions: wild-type (H9, CT2) and SNORD116 deletion lines (H9 smDEL, CT2 smDEL). Black boxes overlaid on the tracks mark RNA fragments captured in chimeras with U3 snoRNA (\u003cstrong\u003eA, E, G\u003c/strong\u003e) or SNORD118 (\u003cstrong\u003eC\u003c/strong\u003e). The red signal indicates Nm enrichment peaks detected by RibOxi-seq2. (\u003cstrong\u003eB, D, F, H\u003c/strong\u003e) Corresponding IntaRNA-predicted base-pairing interactions for the four sites with CLASH overlap. Orange boxes denote regions captured in CLASH hybrids of the target RNA and snoRNA, while purple dashed boxes mark adjacent sequences extended for IntaRNA interaction analysis. Calculated interaction energies (kcal/mol) are shown below each model. Genomic coordinates for the modified nucleoside (Gm) are indicated beneath each panel.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/5c1e2b6b4f736ed12956da1a.jpeg"},{"id":100560064,"identity":"a1a6c863-c115-4966-8530-4d801cbed579","added_by":"auto","created_at":"2026-01-19 08:43:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":814725,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of genes displaying differing Nm positions in CT2 and in small-deletion induced neurons. \u003cstrong\u003eA.\u003c/strong\u003e C12ORF57. Percentage beside the peak denotes modification level quantified by Nm-VAQ, and the corresponding gene expression profiles are shown in red. \u003cstrong\u003eB\u003c/strong\u003e. MIAT. Percentage beside the peak denotes modification level quantified by Nm-VAQ, and the corresponding gene expression profiles are shown in red. \u003cstrong\u003eC.\u003c/strong\u003eAdditional SNORD113/114 targets identified. Base pairing between mRNAs containing Nm sites (highlighted in red) and their respective snoRNAs (SNORD114 and SNORD113). Canonical C/D box sequence motifs are boxed.\u003cstrong\u003e D.\u003c/strong\u003e SNORD116 targets identified.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/7de9a0f15c32aff7520ede9f.jpeg"},{"id":100857672,"identity":"fc85aad2-7332-4845-a1d8-1502de113a33","added_by":"auto","created_at":"2026-01-22 07:18:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5945879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/69e31351-b28e-4bbc-87ce-bcb3c31dbaff.pdf"},{"id":100560614,"identity":"930e8c6f-37da-4b75-be9c-6781fa9cc942","added_by":"auto","created_at":"2026-01-19 08:43:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Data 1.\u003c/strong\u003e Nm sites in 18S and 28S rRNA identified by RibOxi-Seq2 and RiboMeth-Seq, related to Fig. 2.\u003c/p\u003e","description":"","filename":"FileS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/15141c2bf4296e8ea980e17d.xlsx"},{"id":100594824,"identity":"0da97c05-1299-4592-ba67-e35c5fc45df6","added_by":"auto","created_at":"2026-01-19 13:45:25","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":75999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Data 2\u003c/strong\u003e. Nm sites in CT2 and H9 cell lines identified by RibOxi-Seq2, related to Fig. 3.\u003c/p\u003e","description":"","filename":"FileS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/cb29249a06226fe52e1a73a1.xlsx"},{"id":100595142,"identity":"e9997ca4-76aa-4105-b728-2f1f90b5832a","added_by":"auto","created_at":"2026-01-19 13:47:38","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":33853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Data 3.\u003c/strong\u003e Nm-VAQ quantifications, related to Fig. 4, Fig. 6, Supplementary Fig. 2 and Methods.\u003c/p\u003e","description":"","filename":"FileS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/f1b8ed33050687ae1b07cbda.xlsx"},{"id":100559754,"identity":"3e761895-9783-4386-8899-25ddd8565212","added_by":"auto","created_at":"2026-01-19 08:43:36","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":46354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Data 4\u003c/strong\u003e. Nm-VAQ oligos and qPCR-primers, related to Fig. 4, Fig. 6, Supplementary Fig. 2 and Methods.\u003c/p\u003e","description":"","filename":"FileS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/904db34f38fbf1c6d1b119dd.pdf"},{"id":100595268,"identity":"ec62cf36-469d-4b8c-a7a0-34ff015d4d20","added_by":"auto","created_at":"2026-01-19 13:48:05","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1372717,"visible":true,"origin":"","legend":"Supplementary Figures 1,2 3 and 4","description":"","filename":"Suppl.figures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8523796/v1/e6477a5d4fc17ace11f9b79d.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Genome-wide profiling of RNA 2’-O-methylation in neurons and identification of orphan snoRNA targets","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTo date, over 170 distinct RNA modifications have been identified and characterized\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These modifications, which don't alter RNA sequence, play important roles in regulating gene expression and various physiological and pathological processes. Among these RNA modifications, 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylation (Nm) is a widespread RNA modification found in multiple RNA types and species,\u003csup\u003e4\u003c/sup\u003e and it is among the most abundant in the cell owing to numerous sites on rRNAs and small RNAs. 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylation in RNA is thought to primarily function to increase RNA stability, promote RNA folding, facilitate protein binding, and make RNA less susceptible to hydrolysis and cleavage\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe functional impact of Nm is most evident in the regulation of the translational apparatus \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Differential rRNA methylation patterns have been shown to modulate ribosome heterogeneity, effectively \"tuning\" translation to meet specific cellular demands \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Beyond the ribosome, Nm occurs in mRNA, viral RNA, and small RNAs, where it influences alternative splicing, mRNA export, and translational fidelity \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Notably, Nm can sterically hinder the association of RNA-binding proteins (RBPs) and disrupt long-range tertiary interactions that rely on 2\u0026rsquo;-OH hydrogen bonding or divalent metal ion coordination \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and the presence of a 2\u0026rsquo;-O-methyl group can sterically hinder RNA-binding protein association \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe majority of cellular Nm is directed by box C/D snoRNAs, which function as antisense guides within a ribonucleoprotein (snoRNP) complex. Box C/D snoRNAs are defined by conserved sequence motifs\u0026mdash;boxes C/C\u0026rsquo; (RUGAUGA) and D/D\u0026rsquo; (CUGA). The snoRNA utilizes 10\u0026ndash;15 nucleotide antisense elements (ASEs) to base-pair with target transcripts. Canonically, the target nucleotide is positioned exactly five nucleotides upstream of the D or D\u0026rsquo; box. The snoRNA scaffolds a core protein tetramer consisting of Fibrillarin (the methyltransferase), NOP56, NOP58, and SNU13. While snoRNA-mediated methylation is the dominant pathway, independent mechanisms exist, such as the FTSJ3-mediated methylation of the HIV-1 genome, though these non-canonical pathways remain under-characterized \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn humans, 137 out of 267 annotated box C/D snoRNAs\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e have no specified target and are therefore classified as orphan snoRNAs. Possible functions of orphan snoRNAs have been recently reviewed\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. A number of snoRNAs have been shown to target specific sites on mRNA \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and even tRNA\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Also, some have been reported to be involved in alternative splicing\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, alternative polyadenylation \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and other noncanonical processes\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGreater in-depth exploration of Nm biological functions has been limited due to the lack of robust screening tools to detect Nm sites. Mapping Nm positions has proven challenging for many reasons, including resistance to hydrolysis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, the lack of an antibody that can detect Nm and a possibly low stoichiometry of Nm on mRNA. Several high-throughput Nm detection methods have been developed in recent years, including 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003eMe-seq\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, RiboMeth-seq \u003csup\u003e35\u0026ndash;37\u003c/sup\u003e, RibOxi-seq\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, Nm-seq\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, Nm-mut-seq\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and NJU-seq\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. RiboMeth-seq has emerged as the only method that consistently detects all known Nm sites in human rRNAs\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Most importantly, RiboMeth-seq is the only currently available method for the quantitative evaluation of Nm levels at multiple positions, thus providing information on potential Nm variations and providing Nm profiles. However, RiboMeth-seq cannot be used to identify mRNA sites. Recently, Oxford Nanopore Technologies Direct RNA Sequencing (DRS) has been employed by several groups to investigate transcriptome-wide Nm profiles\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, Nanopore-based Nm detection is complicated by signal changes that include read mismatches, deletions, and mutations at neighboring positions. As a result, accurate determination of Nm locations using Nanopore DRS often requires machine learning\u0026ndash;based computational analysis and/or comparison with snoRNA knockout conditions. We have developed RibOxi-seq, which allows the detection of Nm even from small amounts of RNA, including mRNA and non-coding RNA\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Like Nm-seq, this method includes multiple iterative cycles of oxidation/beta elimination-dephosphorylation reactions to sequentially eliminate unmodified nucleotides from 3\u0026rsquo; ends but leave the Nm sites intact. Finally, only RNA fragments with 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylated 3\u0026rsquo; ends can be ligated to linkers for library preparation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Consequently, Nm sites generate a positive signal and not a lack of signal, in contrast to the methods based on the inhibition of reverse transcription (RT) reactions or resistance to hydrolysis. In this study we have further optimized the RibOxi-seq method (now RibOxi-seq2) to make it more sensitive and reproducible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eValidating specific Nm positions has also proved challenging. One sensitive method is RTL-P\u003csup\u003e42\u003c/sup\u003e. When reverse transcription (RT) is performed with low concentrations of dNTPs, the RT enzyme has difficulty incorporating nucleotides opposite a 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylated residue. This leads to premature termination or pausing of cDNA synthesis one nucleotide before the methylated site. However, this method is not reliable for low-abundance RNAs and some structured RNAs\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Recently, a site-specific Nm quantification tool, Nm-VAQ, was described\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The Nm-VAQ (2\u0026rsquo;-O-methylation Validation and Absolute Quantification) method can validate the presence and precisely quantify the stoichiometry of Nm at specific RNA nucleotides by leveraging the property that 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylated RNA is resistant to cleavage by RNase H when it is part of an RNA/DNA hybrid duplex.\u003c/p\u003e \u003cp\u003eSeveral box C/D snoRNAs have been linked to neurodevelopmental disorders. One of the most notable examples is the orphan C/D box snoRNA cluster SNORD116, which is implicated in Prader\u0026ndash;Willi Syndrome (PWS), a rare imprinting disorder affecting approximately 1 in 15,000 newborns\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. PWS results from disruptions of the paternally inherited chromosome 15q11\u0026ndash;q13 region, which harbors\u0026thinsp;~\u0026thinsp;30 tandemly arranged SNORD116 genes\u003csup\u003e\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. This locus is subject to genomic imprinting, with expression dependent on the paternal allele, and deletions in this region are sufficient to cause the disease. Another imprinted snoRNA cluster with neurological relevance is located on chromosome 14q32.2, where the maternally imprinted SNORD112/113/114 genes reside. Deletions affecting this region give rise to Kagami\u0026ndash;Ogata Syndrome, a rare multisystem disorder characterized by skeletal abnormalities and developmental delays including neurodevelopmenlal defects \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo date, there has been no study published on genome-wide Nm profiling in neurons, or in cell models of PWS. To address these knowledge gaps, we have used RibOxi-seq2 to examine isogenic pairs of neurons that only differ in the expression of several orphan box C/D clusters. This allowed us to not only identify neuronal sites of rRNA and small RNA modification but also thousands of mRNA sites, some of which appear to be direct targets of orphan snoRNAs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eHuman embryonic stem cell lines H9 and CT2 were engineered to harbor a doxycycline-inducible neurogenin 2 (NGN2) gene that allows rapid and reproducible differentiation into early postnatal forebrain cortical neurons. Separate isogenic lines were also created in which a small deletion (smDEL) was introduced only on the paternal chromosome 15, removing a cluster of 30 related box C/D snoRNAs (the SNORD116 cluster) that has been implicated in the pathology of Prader-Willi syndrome\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The generation of these cells was recently described\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and NGN2-induced neurons (iNs) from these cell lines, H9, H9-smDEL, CT2, and CT2-smDEL, have been extensively characterized at the transcriptomic level\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Apart from a modest number of overall transcriptomic differences, there are two key features that differentiate the WT and smDEL iNs. First, both the H9-smDEL and CT2-smDEL iNs lack expression of the paternally imprinted SNORD116 cluster of orphan snoRNAs that have been postulated to direct Nms on so far unidentified targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). Second, although originally derived from parent CT2 cells, the CT2-smDEL cells also differ in their expression of a maternally imprinted locus on chromosome 14q32\u003csup\u003e54\u003c/sup\u003e and thus differ from CT2 iNs in the expression of the additional orphan SNORD113/114 clusters, comprising 9 and 31 copies, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Importantly, both SNORD113/114 and SNORD116 snoRNAs are expressed in the brain. Thus, the CT2-smDEL iNs can serve as useful knockout lines for several specific and physiologically relevant subsets of orphan snoRNAs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRibosomal RNA modifications in stem cells and neurons\u003c/b\u003e. Given that the relationship between Nm and neuronal disorders remains poorly understood, we sought to gain detailed insights into Nm patterns in an isogenic pair of neuronal populations. RNAs were subjected to RibOxi-seq2 and sites displayed on the UCSC Genome Browser. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-f shows representative profiles of 18S and 28S rRNA for CT2, CT2-smDEL, H9, and H9-smDEL iNs. Every peak observed represents an annotated or validated Nm site (\u003cb\u003eSupplementary Data 1\u003c/b\u003e). Notably, the peaks are qualitatively consistent between the four cell samples, revealing not only the reproducibility of the RibOxi-seq2 method but also reinforcing the fact that SNORD116 snoRNAs are true orphans as they appear not to target any rRNA Nm. An alternative method, RiboMeth-seq, was next used to corroborate and extend these results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f). This method has been shown to be highly sensitive and quantitative for rRNA Nm mapping. RiboMeth-seq identified 44 Nm sites in 18S rRNA (3 Nm sites were validated using the site-specific Nm quantification tool, Nm-VAQ\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e) and 67 Nm sites in 28S rRNA, while RibOxi-seq2 data identified 42 Nm sites in 18S rRNA and 61 Nm sites in 28S rRNA (\u003cb\u003eSupplementary Data 1\u003c/b\u003e). In 18S rRNA, using both RibOxi-seq2 and RiboMeth-seq, we identified two Nm sites, Um966 and Um1445, that are known to be highly modified as pseudouridines (Ψ), suggesting the likelihood of double modification (Ψm) at these sites (for this cellular context) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Likewise, in 28S rRNA, U3818 is known to be modified as Ψm3818, and we also see it as Nm in RibOxi-seq2 tracks and validated this by Nm-VAQ. One weakness of RibOxi-seq2 is that if there are consecutive Nm sites, only the most 3\u0026rsquo; site is observed, and this weakness accounts for most of the discrepancy between the two methods. Thus, 61/63 detectable sites in 28S rRNA were observed by RibOxi-seq2. In order to compare rRNA modifications between pluripotent cells and neurons, we also performed RiboMeth-seq on undifferentiated H9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d \u003cb\u003eand Supplementary Data 1\u003c/b\u003e.) Nm profiles between H9 cells and H9 iNs were essentially the same except that 18S U354 is unmethylated in stem cells and largely methylated in iNs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f). These data reveal dynamic ribosomal RNA patterns across different cell lines, suggesting that specific modification levels may contribute to cell-type-specific differentiation. Interestingly, 18S Um354 has previously been associated with differentiation and is directed by SNORD90\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt was recently reported that SNORD113/114 snoRNAs may maintain hematopoietic stem cell self-renewal at least in part by altering rRNA Nm levels\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Here we observe no 18S or 28S rRNA Nm differences between CT2 and CT2 smDEL iNs, which differ in expression of SNORD113/114. Nm profiles were also analyzed in 5.8S rRNA species and no differences in Nm were detected.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSmall RNA modifications in neurons\u003c/b\u003e. RibOxi-seq revealed Nm sites in a number of tRNAs. For example, the first nucleotide in the anticodon loop was 2\u0026rsquo;-O-methylated in tRNA-Gly-CCC-2-2 (Um31), tRNA-Val-CAC-1-4 (Cm31), tRNA-Ala-AGC-2-1 (Um31) and tRNA-Cys-GCA-2-2 (Cm31) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg \u003cb\u003eand Supplementary Data 1\u003c/b\u003e). This position is highly conserved compared to the Nm at nucleotide 32 reported in tRNA-Phe, and has been shown to be enzymatically catalyzed by the protein FTSJ1 in human (yeast homolog Trm7), or mediated by SNORD97 or SNORD133 in a snoRNA-dependent fashion\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Since these Nm in the anticodon region were not mediated by SNORD113/114/116 snoRNAs, no differences were observed between H9/CT2 wild type and smDEL iNs. Further, Um38 in tRNA-Gly-CCC-2-2 and Cm39 in tRNA-Glu-CTC-1-7 were identified at the first nucleotide following the anticodon loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg \u003cb\u003eand Supplementary Data 1\u003c/b\u003e). These modifications are conserved and correspond to previously annotated (Um/Gm/Ψm39) \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The Nm at position 39 was reported to be catalyzed by a snoRNP complex\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. As expected, m5Um (2\u0026rsquo;-O-methyl-5-methyluridine) residues located at the first nucleotide in the T-loop region were also reproducibly captured, including tRNA-Lys-CTT-2-5 (m5Um-54), tRNA-Leu-AAG-2-4 (m5Um-63), tRNA-Gly-GCC-2-4 (m5Um-52), tRNA-Glu-CTC-1-7 (m5Um-53), and tRNA-Asn-GTT-1-1 (m5Um-55) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg \u003cb\u003eand Supplementary Data 1\u003c/b\u003e). The m5Um identity and position match previous mass spectrometry results\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, indicating that the m5Um modification also resists sodium periodate treatment, and RibOxi-seq2 cannot clearly differentiate between Nm and m5Um modifications since both are 2\u0026rsquo;-O-methylated nucleotides.\u003c/p\u003e \u003cp\u003eIn addition, reproducible Nm sites were identified in U1 (Am70), U4 (Am65), U5 (Gm37, Um41, Cm45), and U6 (Am70, Cm77) snRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u003cb\u003e).\u003c/b\u003e The sites extend our recent work using RibOxi-seq to map Nm sites on U6, supporting the robustness of RibOxi-seq in mapping Nm even in small RNA species\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Nm modifications in U2 snRNA were not detected, most likely due to the RNA fragmentation procedure failing to enrich small U2 snRNA fragments. Interestingly, we found a novel Nm site in 7SK RNA, Gm240 (\u003cb\u003eSupplementary Data 1\u003c/b\u003e). This site is two nucleotides downstream of A238 which has been reported to be modified as m6A\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. As no Nm sites have been reported previously in 7SK RNA, we don\u0026rsquo;t yet know whether this modification occurs in cells other than neurons. Collectively, the RibOxi-seq2 method faithfully identified Nm sites in rRNA, tRNA, and snRNA. As expected, no Nm level changes were found in small RNAs between wild type and smDEL iNs. Together, these results suggest that RibOxi-seq2 may prove useful for mapping many Nm sites even in small RNAs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWidespread and robust modifications in mRNAs\u003c/b\u003e. A recent study identified thousands of Nm sites in mRNAs of human and mouse cell lines using NJU-seq and validated a number of them using Nm-VAQ\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Of thousands of Nm sites identified, about a third were conserved between different cell types. These authors revealed a broad distribution of Nm sites on mRNAs and observed that in their system most validated Nm sites were methylated at ratios from 1% to 30%. Here using RibOxi-seq2 we have also identified thousands of likely Nm sites within mRNAs and lncRNAs, a number of which were validated by Nm-VAQ (\u003cb\u003eSupplementary Data 2\u003c/b\u003e). Importantly, this approach yielded consistent Nm peaks across H9, CT2, and smDEL cell lines, providing strong evidence for the robustness of the method (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Owing to a higher sequencing depth in the CT2 samples, we could identify more sites in the CT2 data (989 Nm sites with Nm score 1000 or higher) than in the H9 data (736 Nm sites with Nm score 1000 or higher). When we selected the top 500 sites between both cell types, it was clear that a majority of identified Nm sites are shared between the two parental cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Also, for both H9 and CT2 iNs, Nm profiles were almost identical between the WT and smDEL cells, again highlighting the reproducibility of RibOxi-seq2. Overall, about 40% of Nm sites were in 3\u0026rsquo;-UTRs, 5% in 5\u0026rsquo;-UTRs, and the rest in coding exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). While most are shared, some Nm sites were observed only in H9 iNs or CT2 iNs. One example of this is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed. UCHL1 exhibits a strong site in H9 iNs but not in CT2 iNs. Also, the major TOP1 Nm site is in exon 9 in H9 cells but in exon 12 in CT2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Gene ontology analysis revealed that Nm sites are broadly distributed in biological processes, with no clear preference for any pathway (not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we employed Nm-VAQ to validate the modification levels at several mRNA Nm sites. Results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Surprisingly, a number of neuronal mRNAs exhibited very high degrees of methylation, in some cases approaching complete modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). For instance, two sites in ACTB exons 4 and 5 were methylated at 97% and 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), respectively (N.B., RibOxi-seq2 Nm browser peaks as displayed are not quantitative); a site in the 5\u0026prime;-UTR translational control element of TPT1 was modified at 88% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec); and a site in SNCG exon 3 showed 100% modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In addition, we detected a site in NUDT21 exon 1 at 50% (\u003cb\u003eSupplementary Data 2\u003c/b\u003e) and another in the 3\u0026prime;-UTR of FAM171A1 at 43% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), indicating that not all sites are heavily modified. By contrast, certain Nm sites displayed much lower modification levels. For example, a site in MT-CO1 was modified at only 6.7% and one in FGF13 at 24% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee \u003cb\u003eand Supplementary Data 2\u003c/b\u003e). Taken together, these results demonstrate that, at least in iNs, mRNA Nm modifications occur across a wide dynamic range, from nearly absent to fully saturated. Additionally, the Nm sites we identified in neuronal cell lines did not overlap with the mRNA Nm sites previously reported in HeLa cells\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, suggesting dynamic and/or cell line\u0026ndash;specific Nm regulation and variation, or methodologic differences.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLack of canonical snoRNA targeting of most mRNA sites.\u003c/b\u003e While all rRNA and most snRNA Nm sites are known to be the targets of box C/D snoRNAs or scaRNAs, this has not been shown yet for most mRNA sites. Our large number of shared Nm sites from our four iN cell lines offered the possibility to test this possibility. We selected CT2 sites with Nm scores above 500 (over 500 sites) and analyzed them by SnoScan\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e for canonical complementarity to known snoRNAs. Thus, successful hits must exhibit significant complementarity to snoRNAs with the Nm site positioned 5 nucleotides upstream of the snoRNA box D or box D\u0026rsquo; (CUGA) motif. Surprisingly, almost none of the abundant shared sites satisfied these criteria. Thus, overall, the vast majority of Nm sites on this list could not be assigned to canonical snoRNA interactions. However, some sites not shared between cells and of lower expression levels could be matched (see below). We next explored the possibility that noncanonical snoRNA-mRNA interactions may play a role in some Nm modifications. While the abundant U3, U8 and U13 snoRNAs have not been previously associated with methyltransferase activity, a recent report studying mRNA-snoRNA physical interactions (sno-KARR-seq) revealed that a high proportion of mRNAs apparently associated with U3 snoRNA, although not necessarily involving canonical basepairing\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Further, those authors reported that U3 snoRNA knockdown led to a significant decrease in overall cellular Nm levels. Another group investigating crosslinking of snoRNA-mRNA hybrids also noted a very high number of U3-mRNA interactions\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Together, these studies raised the possibility that this snoRNA may have a role in the cell other than in rRNA processing. Interestingly, the sequences around many Nm sites could be aligned with U3, U8 (SNORD118) or U13 snoRNAs by searching for antisense complementarity with snoRNAs catalogues in the snoDB\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Comparison of minimum folding energy (MFE) across snoRNAs and RNA regions encompassing each Nm site (\u0026plusmn;\u0026thinsp;7 nt) (\u003cb\u003eSupplementary Fig.\u0026nbsp;3)\u003c/b\u003e revealed a possible connection between U3 snoRNA and many Nm sites.\u003c/p\u003e \u003cp\u003eU3 snoRNA participates in the first stage of rRNA processing\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e and has been reported to exist in multiple molecular complexes, some containing the canonical box C/D components (Fibrillarin, NOP56, NOP58, and SNU13/15.5K). U3 snoRNA complexes have also been reported to shuttle between the nucleus and cytoplasm\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Consistent with a model in which noncanonical snoRNAs directly guide a subset of the Nm sites detected in our RibOxi-seq dataset, several of the most prominent Nm peaks overlapped with snoRNA\u0026ndash;mRNA chimeras captured by U3 or U8 (SNORD118) in published 293T CLASH data\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Four representative examples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and additional overlaps of CLASH fragments with Nm peaks are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e. For GUK1, a discrete Nm peak in both H9 and CT2 cells aligns exactly with a U3-derived CLASH fragment, and IntaRNA\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e predicts a stable U3\u0026ndash;GUK1 interaction spanning the modified nucleotide with an interaction energy of \u0026minus;\u0026thinsp;10.5 kcal/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). A similar pattern is observed at RPL7A, where a strong Nm peak corresponds to a hybrid captured with SNORD118, again supported by a favorable predicted interaction (\u0026ndash;9.9 kcal/mol) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Two additional mRNAs, AZIN1 and KMT2A, show the same concordance: each features a sharp Nm peak detected by RibOxi-seq2 that overlaps with U3-captured CLASH reads, and each forms a thermodynamically stable interaction with U3 in IntaRNA modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d). Notably, for all four transcripts, the strongest predicted base pairing occurs directly over, or immediately adjacent to the Nm-modified position, although these interactions lack the canonical alignment with a recognizable D-box element. Together (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003eand Supplementary Data 4\u003c/b\u003e), these examples are consistent with a model that a subset of mRNAs may acquire 2\u0026prime;-O-methylation through direct interaction with snoRNAs such as U3 and SNORD118, via a noncanonical mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTargets of orphan SNORD113/114 snoRNAs\u003c/b\u003e. Since CT2 iNs and CT2-smDEL iNs differ in expression of the orphan SNORD113/114 snoRNA clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), we asked whether these cells could be used to identify novel targets for these specific snoRNAs. Our approach was to compare Nm profiles, searching for Nm signals in CT2 iNs that are missing in CT2-smDEL iNs as well as in both H9 iNs and H9-smDEL iNs, which also lack expression of the SNORD113/114 region. This was facilitated by the reproducibility of our data. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea shows a genomic region witha strong peak in the C12orf57 3\u0026rsquo;-UTR in CT2 iNs that is completely lacking in the CT2-smDEL data. This site is complementary to SNORD114 with the Nm positioned 5 nucleotides upstream of the box D element, consistent with this site being a canonical target. Nm-VAQ revealed that this site is modified in 24% of the transcripts. Another (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) in the lncRNA MIAT is seen in 94% of transcripts. A similar strategy allowed us to identify additional SNORD113 and SNORD114 targets including PCDH10, SLC22A17, ANKRD13B, SMG5, PARP6, DDX17 and a number of noncoding transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Importantly, these genes are closely linked to fundamental processes in brain development, and their dysfunction is consistent with mechanistic basis for the onset of neurological disorders such as Kagami-Ogata syndrome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTargets of orphan SNORD116 snoRNAs.\u003c/b\u003e Since lack of expression of SNORD116 snoRNAs has been related to Prader-Willi syndrome, the smDEL cell lines with SNORD116 cluster deletions represent a valuable resource to investigate the molecular basis of the disease. In order to qualify as potential canonical sites, there needed to be an Nm signal in both H9 and CT2 iNs but no signal at the same position in both H9-smDEL and CT2-smDEL iNs. Also, the Nm sites needed to be complementary to SNORD116 with the Nm site positioned 5 nucleotides upstream of a box D or D\u0026rsquo; element. While we found a number of SNORD113/114 targets, we were only able to identify two potential SNORD116 targets. However, the two targets identified are of considerable interest. These are NLGN3 and DGKK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). For each of these mRNAs, there is extensive basepairing with multiple SNORD116 species and the potential Nm sites are 5 nucleotides upstream of the box D element. However, as PWS pathology is closely connected to lack of expression of SNORD116s, our failure to identify numerous robust SNORD116 targets was unexpected. This could be for several reasons. First, some physiological SNORD116 targets may not be expressed well in our iNs. Since PWS is generally thought to be primarily a disorder affecting the hypothalamus, some targets most abundantly expressed there could be poorly expressed in iNs. Second, our findings could indicate that these snoRNAs, which are highly abundant in neurons, may have additional important functions apart from guiding 2\u0026rsquo;-O-methylation. Third, some PWS pathology may be connected not directly to SNORD116 snoRNAs, but to expression of other noncoding transcripts expressed from this genomic region\u003csup\u003e\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we have used RibOxi-seq2 to generate the first comprehensive picture of the Nm landscape in neurons. Sequencing was carried out on two isogenic pairs of NGN2-induced neuronal cultures derived from embryonic stem cell lines. Each isogenic pair included one wild-type cell line and another harboring a small deletion of 30 related tandem orphan snoRNAs (SNORD116s) from the paternal chr15q11-q13 region, whose loss of expression is associated with Prader-Willi syndrome. The CT2-based isogenic pair also differed in expression of the orphan SNORD113/114 clusters from the imprinted chr14q32.2 region. Thousands of shared Nm sites were identified in these cells, in both coding and noncoding RNAs. The most abundant cellular Nm modifications occur in rRNA. In rRNA, RibOxi-seq2 identified almost all annotated Nm sites but is limited by two technical issues. First, when two Nm sites exist in tandem, RibOxi-seq2 can only see the distal site, except when this site is not fully modified. This led to an inability to monitor several sites in both 18S and 28S rRNA. Second, since RibOxi-seq2 is qualitative and not quantitative, sites modified at a low level even in abundant RNAs such as rRNA can lead to some Nm peaks that are not dramatically higher than background signals. Owing to these issues, we further analyzed ribosomal Nm profiles using RiboMeth-seq, which does not suffer from these drawbacks. Results showed not only that RibOxi-seq2 is highly efficient at identifying rRNA Nm sites, but also that ribosomal RNA in iNs has a distinct modification signature compared to that in embryonic stem cells. In particular, Um354 in neuronal 18S rRNA was seen by both methods and has been reported to be more highly expressed in differentiated cells and directed by SNORD90\u003csup\u003e55\u003c/sup\u003e. This raises the question of how a specific rRNA Nm profile affects translational efficiency or regulation in neurons. Might it affect the translation not only of neuronal mRNAs, but also those harboring neuron-specific Nms? Apart from rRNA, we also observed a number of reported Nm sites in other abundant noncoding RNAs such as snRNAs and tRNAs, showing that RibOxi-seq2 has the potential to monitor such sites in RNA samples. We also identified and validated a novel Nm site in 7SK RNA.\u003c/p\u003e \u003cp\u003eOwing to its ability to detect Nm sites even in low-abundance RNAs, RibOxi-seq identified thousands of Nm sites in hundreds of mRNAs and lncRNAs. Importantly, a large fraction of these sites appeared in all four iN cultures, while only a small fraction of Nm sites appeared to be cell- or parent-specific. We don\u0026rsquo;t yet know the basis of specificity, but we hypothesize that the shared Nm sites include a neuronal signature of genome-wide 2\u0026rsquo;-O-methylation. While most Nms are shared between the different neuron samples, some are cell-specific. Nm sites in mRNAs are broadly distributed both in functional categories and in regions of internal RNA modification. Thus, about 5% are in 5\u0026rsquo;-UTRs, 40% in 3\u0026rsquo;-UTRs, and the rest in coding exons. While some mRNAs may harbor sites that are partially modified, other mRNAs have Nm sites that are almost fully modified. Nm-VAQ revealed that a number of sites in abundant mRNAs are present at almost 100% modification. Such high levels of modification have also been reported recently by others\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, though most sites identified in this study do not match the positions reported in that work and may thus represent important cell-specific RNA modifications or differences in methodology.\u003c/p\u003e \u003cp\u003eWhat targets most mRNA Nm sites? In our studies, we were surprised to discover that we could not match the great majority of mRNA Nm sites to canonical RNA-snoRNA interactions. This suggested that such modifications may occur via noncanonical interactions, or that many may be directed via recruitment of other activities such as the methyltransferase FTSJ3. Intriguingly, however, we observed that many sites exhibit complementarity to abundant but noncanonical snoRNAs such as U3, U13, and U8 (SNORD118). Do these snoRNAs and their associated proteins harbor methyltransferase activities or have the ability to recruit them? U3 has been reported to exist in multiple distinct snoRNP complexes, and U3 as well as some of its protein components have been shown to shuttle between the nucleus and cytoplasm\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Our findings with U3 are consistent with the recent report of abundant U3-mRNA interactions using the sno-KARR-seq RNA-RNA interaction method\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Importantly, those authors observed that knockdown of U3 led to a reduction in overall levels of mRNA Nm modification. Another group also reported a very high number of U3-mRNA interactions\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Together with our CLASH results, these findings lead us to hypothesize that U3, and perhaps also U13 and U8, play an important role in mRNA Nm modification in addition to a role in rRNA maturation.\u003c/p\u003e \u003cp\u003eWhile Nm sites in abundant mRNAs could not be assigned to snoRNA interactions following established rules, our results support the assertion that some Nm peaks do represent canonical snoRNA-mRNA interactions. This possibility is supported by the identification of a number of sites that appear to be canonical targets of previously annotated orphan snoRNAs, including members of the SNORD113, 114, and 116 families. Our data represent the first identification of likely targets of these snoRNAs. Most of these targets are in RNAs that are connected to neurodevelopmental disorders.\u003c/p\u003e \u003cp\u003eDeletion of the SNORD113/114 region (chr14q32.2) leads to the rare multi-symptom disorder Kagami-Ogata syndrome\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Specific expression patterns of SNORD113/114 snoRNAs in the brain and strong associations with neurodevelopmental disorders point to crucial and specialized roles in brain development, function, and potentially in the pathogenesis of neurological conditions. SNORD113-6 has been reported to methylate tRNA\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e but our data suggest a possible additional role in mRNA modification. Novel mRNA targets of SNORD113 that we have identified here include PARP6 and DDX17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). PARP6 is a regulator of hippocampal dendritic morphogenesis\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. DDX17 interacts with and co-regulates the transcriptional repressor element 1-silencing transcription factor (REST), which is a critical regulator of neuronal differentiation, suppressing neuronal gene expression in progenitor cells\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSNORD114 targets also shed light on potential roles in neuronal development. C12orf57 has been reported to be a critical gene involved in brain development, particularly the formation of the corpus callosum and the regulation of synaptic function. It is also involved in neurodevelopment and is a regulator of synaptic AMPA currents and excitatory neuronal homeostasis\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Mutations in this gene are associated with developmental brain abnormality\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. MIATis a lncRNA associated with neuronal development\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. PCHD10, a cadherin-related neuronal receptor, is associated with autism and neuropsychiatric disorders\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e,\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. SLC22A17 (Solute Carrier Family 22 Member 17), also known as the lipocalin-2 receptor, has emerged as a significant player in the brain, particularly in the context of neuroinflammation and neurodegeneration. While initially identified for its role in organic cation transport and iron homeostasis, recent research has highlighted its critical involvement in the blood-brain barrier (BBB) integrity and cell death pathways, especially after stroke\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. ANKRD13B is a ubiquitin-binding protein that is widely expressed in the brain. Finally, it is noteworthy that SNORD114 appears to target a number of noncoding transcripts, including lncRNAs and miRNA host genes. PKD1P6-NPIPP1 is a cell-cycle related lncRNA and has been implicated as a prognostic marker for hepatocellular carcinoma\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Further studies are warranted to investigate the functional ramifications of these Nm modifications.\u003c/p\u003e \u003cp\u003eWe also found two potential SNORD116 targets, DGKK and NLGN3, and these genes have functions consistent with roles in PWS. NLGN3 (neuroligin 3) is a crucial postsynaptic cell adhesion molecule that plays a pivotal role in organizing and stabilizing synapses and which is linked to autism\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. It is also a key regulator of gonadotropin-releasing hormone deficiency and is connected to hypogonadism\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. DGKK (diacylglycerol kinase kappa) converts diacylglycerol to phosphatidic acid, regulating the balance between these two important signaling lipids in the brain. These lipids are involved in neurotransmitter signaling, synaptic plasticity, and overall neuronal communication\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. DGKK has been reported to be a (and possibly the) primary target of the Fragile X-related protein FMRP\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. Interestingly, both NLGN3 and DGKK are translationally upregulated by FMRP\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e so the translational consequences of Nm modifications in their mRNAs clearly warrant future study. Thus, both of the potential SNORD116 targets identified here may contribute to the developmental, neurological, and behavioral features of PWS. In fact, among others, both of these genes were hypothesized as possible PWS targets in previous work\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, we note that the isogenic cell models described here may prove useful in additional studies of Nm modifications and functions. As these cells are embryonic stem cells, they can be differentiated into a multitude of additional cell and organ types, and their differences in orphan snoRNA expression can be exploited to examine the roles of SNORD113, SNORD114, and SNORD116 box C/D snoRNAs in multiple tissue types, both at transcriptomic and targeted Nm levels.\u003c/p\u003e"},{"header":"Limitations of the study","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eH9 and CT2 cells provide relevant physiological models for investigating PWS and CT2 cells provide a useful model for the study of functions of SNORD113 and SNORD114; however, Nm sites present in low abundance species may be overlooked by the RiboXi-seq2 method. Additionally, as both cell lines are derived from female donors, future studies employing male-derived cell lines will be important.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePWS is thought to be largely a disorder connected to the hypothalamus, and SNORD113/114 targets, which may exist in numerous other cell types. By differentiating into different cell and organ types, the CT2/CT2 smDEL isogenic cell pair of human ESCs used here may offer the potential to gain even further insights into the functions of SNORD113, SNORD114, and SNORD116.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eWhether Nm modifications occur co-transcriptionally or post-transcriptionally; or primarily in the nucleus or in the cytoplasm has not been firmly established. This issue is particularly relevant for U3, which has been reported to be at least partially localized to the cytoplasm. Further studies will be necessary to clarify the mechanisms of U3/U8 (SNORD118)/U13 to mRNA Nm targets.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":" Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCulture and differentiation were carried out as described previously \u003csup\u003e53\u003c/sup\u003e. CT2 and H9 human embryonic stem cells (hESCs) including SNORD116 deletion variants were cultured under feeder-free conditions using Matrigel-coated (Corning, #354277) 100-mm dishes and maintained in mTeSR Plus (STEMCELL Technologies, #100-0276) in a humidified atmosphere at 37°C with 5% CO₂. The culture medium was replaced daily, and cells were passaged approximately every 4–5 days upon reaching 80–90% confluency. For passaging, the medium was removed, and dishes were treated with 0.5 mM EDTA (Invitrogen, #15575020) in PBS, followed by a 2-minute incubation at 37°C. The EDTA solution was then aspirated, and fresh mTeSR Plus medium was added. The cell aggregates were carefully detached using either a StemPro EZPassage Tool (Gibco, #23181010) or by gently scratching the dish with a glass pipette. The passage ratio was maintained at approximately 7–10 to ensure optimal growth.\u003c/p\u003e\n\u003cp\u003eWhen the hESCs reached 70–80% confluency, cells were prepared for differentiation. First, any differentiated cells were manually removed, and the 100-mm dish was rinsed with PBS. The cells were then treated with Accutase (Millipore, #SCR005) and incubated for 2 minutes at 37°C. After incubation, 10 mL of basal media was added to the cell suspension, which was transferred to a 50-mL conical tube and centrifuged at 1200 rpm for 3 minutes. The supernatant was aspirated, and the pellet was resuspended in 1 mL of Induction Media (IM). The cells were singularized by pipetting up and down 3–5 times using a 1-mL pipette. An additional 9 mL of IM was then added to the suspension.\u003c/p\u003e\n\u003cp\u003eThe Induction Medium (IM) was prepared by supplementing DMEM/F12 (with HEPES) (Gibco, #11330032) with 1X N-2 Supplement (Gibco, #17502048), 1X MEM Non-Essential Amino Acids (Gibco, #11140050), and 1X GlutaMAX Supplement (Gibco, #35050061). \u003c/p\u003e\n\u003cp\u003eFor differentiation, 4 million cells were plated in a Matrigel-coated (Corning, #354277) 100-mm dish and cultured in IM supplemented with 2 μM Doxycycline Hydrochloride (Fisher Scientific, #BP26535). The cells were fed daily with IM containing 2 μM doxycycline hydrochloride for four days.\u003c/p\u003e\n\u003cp\u003eOn day 4 of differentiation, the cells were singularized again using Accutase as described above. The cell pellet was resuspended in Cortical Media (CM), which was prepared by mixing equal volumes of DMEM/F12 with HEPES and Neurobasal Medium (Gibco, #21103049) and supplementing with 1X B27 Supplement (Gibco, #17504044), 10 ng/mL Recombinant Human BDNF Protein (R\u0026amp;D Systems, #248-BD), 10 ng/mL Recombinant Human GDNF Protein (R\u0026amp;D Systems, #212-GD), 10 ng/mL Human NT-3 Recombinant Protein (PeproTech, #450-03), and 1 μg/mL Laminin Mouse Protein (Gibco, #23017015). \u003c/p\u003e\n\u003cp\u003eCells were plated at a density of 13 million cells per 100-mm dish in CM supplemented with 10 μM ROCK inhibitor Y-27632 dihydrochloride (Tocris, #1254). A complete medium change with CM was performed the following day, and the media was subsequently changed daily until collection on day 11.\u003c/p\u003e\n\u003cp\u003eThe 100-mm dishes were pre-coated with 100 μg/mL Poly-D-lysine hydrobromide (PDL, Millipore Sigma, #P0899) and 5 μg/mL laminin. Initially, 6 mL of the 100 μg/mL PDL working solution was applied to each dish and incubated overnight to ensure adequate coating. The next day, the dishes were thoroughly washed twice with PBS to ensure they remained hydrated. Subsequently, 6 mL of the 5 μg/mL laminin working solution was added to the PDL-coated dishes, which were then incubated overnight to prepare them for neuron differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using the TRIzol reagent protocol. Briefly, cells were washed with PBS, pelleted by centrifugation, and lysed in TRIzol reagent (Invitrogen, #15596026). After incubation at room temperature for 5 minutes, 0.2 mL of chloroform (Invitrogen, #288306) per mL of TRIzol was added, followed by vigorous shaking and centrifugation at 12,000 × g for 15 minutes at 4°C. The aqueous phase containing RNA was collected, mixed with 0.5 mL of isopropanol (Invitrogen, # I9030) per mL of TRIzol, incubated for 10 minutes, and centrifuged at 12,000 × g for 10 minutes at 4°C. The resulting RNA pellet was washed with 75% ethanol, air-dried, and resuspended in RNase-free water. RNA cleanup and DNase I treatment were performed using the RNA Clean \u0026amp; Concentrator-25 (Zymo Research, #R1017) according to the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Ethanol Precipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 100 µL of RNA solution, 1 µL of Linear Acrylamide (250–500X; Invitrogen, AM9520) and 250 µL of ice-cold 100% ethanol (2.5–3.0 volumes) were added. The mixture was thoroughly mixed and stored at −80°C for 1 hour or overnight at −20°C to allow RNA precipitation. The precipitated RNA was recovered by centrifugation at 14,000 × g for 20 minutes at 4°C. The supernatant was removed, and the RNA pellet was washed with 0.5 mL of ice-cold 75% ethanol, followed by centrifugation at 10,000 × g for 5 minutes at 4°C. Residual ethanol was carefully removed using a 20-µL pipette after a brief spin-down, and the pellet was air-dried on ice for 5–10 minutes before being resuspended in nuclease-free water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Ethanol Precipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 100 µL of DNA solution, 10 µL of 3M Sodium Acetate (Invitrogen, #AM9740), 1 µL of GlycoBlue Coprecipitant (Invitrogen, #AM9515), and 250 µL of ice-cold 100% ethanol (2.5–3.0 volumes) were added. The solution was thoroughly mixed and stored overnight at −20°C to allow DNA precipitation. The DNA was recovered by centrifugation at 14,000 × g for 20 minutes at 4°C. The supernatant was carefully removed, and the DNA pellet was washed with 0.5 mL of ice-cold 75% ethanol, followed by centrifugation at 10,000 × g for 5 minutes at 4°C. Residual ethanol was carefully removed using a 20-µL pipette after a brief spin-down, and the pellet was air-dried on ice for 5–10 minutes before being resuspended in nuclease-free water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRNA Isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA was isolated from 1-1.5 mg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, #E7490L) following the manufacturer's protocol. After the isolation procedure, the purified mRNA was collected by transferring 88 μL of the supernatant to a 1.5 mL DNA LoBind Tube (Eppendorf, #0030108051). The integrity of the isolated mRNA was assessed using RNA ScreenTape and RNA ScreenTape Sample Buffer (Agilent, #5067-5576 and #5067-5577).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRibOxi-seq2 Methods \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese were essentially as described \u003csup\u003e39\u003c/sup\u003e but with a number of modifications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Fragmentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 0.25 U/μL benzonase working solution was prepared by mixing 100 μL of 10× benzonase buffer, 899 μL of nuclease-free water, and 1 μL of 250 U/μL Benzonase Nuclease (Millipore Sigma, #E8263). For fragmentation, 88 μL of 10–20 μg mRNA was heated at 95°C for 3 minutes in a ThermoMixer C (Eppendorf) and immediately placed on ice for 3 minutes. Subsequently, 10 μL of 10× benzonase buffer and 2 μL of the benzonase working solution were added to the mRNA solution. The mixture was vortexed, briefly centrifuged, and immediately incubated on ice for 80 minutes. The digestion time requires optimization, particularly for different cell lines and different batches of benzonase. \u003c/p\u003e\n\u003cp\u003ePost-fragmentation: 100 μL of nuclease-free water and 200 μL of Acid-Phenol:Chloroform (pH 4.5; Invitrogen, #AM9722) were added to the sample. The mixture was vortexed for 15–20 seconds, incubated at room temperature for 2 minutes, and centrifuged at 18,000 × g for 10 minutes at 4°C. The aqueous phase (180–190 μL) was carefully transferred to a new tube. RNA ethanol precipitation was performed as described above. The final RNA was suspended in 64 μL of nuclease-free water. RNA quality was assessed using 1 μL of the sample, confirming the presence of a sharp peak between 25–200 nucleotides, skewing to the right.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Oxidation, β-Elimination, and Dephosphorylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 200 mM Sodium meta-periodate (NaIO4; Thermo Fisher, #20504) solution was prepared by dissolving 42.78 mg of NaIO4 in 1 mL of nuclease-free water. The solution was protected from light, kept on ice, and used the same day. For the oxidation reaction, 64 μL of fragmented RNA was mixed with 8 μL of oxidation-elimination buffer (pH 8.5) and 8 μL of NaIO4 (200 mM), resulting in a final volume of 80 μL. The mixture was vortexed, briefly centrifuged, and incubated at 37°C with shaking (350 rpm) for 45 minutes in a ThermoMixer C (Eppendorf). Following oxidation, the RNA was purified using ethanol precipitation, and cleaned up using Micro Bio-Spin P-6 Gel Column (Bio-Rad, #7326221), with the RNA eluted in 84 μL of nuclease-free water.\u003c/p\u003e\n\u003cp\u003eThe oxidation-elimination buffer was prepared from 2M L-Lysine monohydrochloride (Sigma-Aldrich, #L5626) in nuclease-free water. The pH was adjusted to 8.5 using 2 M NaOH, and the solution was filtered through a 0.22 μm filter.\u003c/p\u003e\n\u003cp\u003eFor the dephosphorylation reaction, 84 μL of oxidized RNA was mixed with 10 μL of rCutSmart buffer (10×, New England Biolabs, #B6004S), 2 μL of RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019), and 4 μL of Quick CIP (5 U/μL, New England Biolabs, #M0525S), resulting in a final volume of 100 μL. The reaction was incubated at 37°C for 10 minutes, followed by heat inactivation at 80°C for 2 minutes. The RNA was subsequently purified using ethanol precipitation.\u003c/p\u003e\n\u003cp\u003eThe entire oxidation, β-elimination, and dephosphorylation process was repeated three additional times. Subsequently, RNA cleanup was performed using the Zymo RNA Clean \u0026amp; Concentrator-25 (Zymo Research, #R1017). The final RNA was eluted in 66 μL of nuclease-free water.\u003c/p\u003e\n\u003cp\u003eFurther dephosphorylation reactions involved two stages. In stage I, 66 μL of oxidized RNA was mixed with 8 μL of T4 PNK buffer (10×, pH 6.0), 4 μL of T4 polynucleotide kinase (10 U/μL, New England Biolabs, #M0201), and 2 μL of RNaseOUT inhibitor, resulting in a total volume of 80 μL. The mixture was incubated at 37°C for 3 hours.\u003c/p\u003e\n\u003cp\u003eIn stage II, 80 μL of oxidized RNA from the first reaction was combined with 10 μL of T4 PNK buffer (10×, pH 7.6), 4 μL of T4 Polynucleotide Kinase (10 U/μL, NEB, #M0201S), 20 μL of 10 mM Adenosine 5'-Triphosphate (ATP; New England Biolabs, #P0756S), and 66 μL of nuclease-free water to a final volume of 180 μL. This reaction was incubated at 37°C for 1 hour and inactivated with the addition of 2 μL of 0.5 M EDTA. The RNA was purified by ethanol precipitation and resuspended in 70 μL of oxidation-only buffer. The Oxidation-only buffer was prepared with 4.375mM Sodium Tetraborate Decahydrate (Fisher Scientific, #BP175-500), 50mM Boric Acid (Fisher Scientific, #A74-1), and nuclease-free water. The pH was adjusted to 8.6 using 2 M NaOH, and the solution was filtered through a 0.22 µm filter before use.\u003c/p\u003e\n\u003cp\u003eAn additional oxidation reaction was performed by combining 70 μL of oxidized RNA with 10 μL of NaIO4 solution (200 mM) to a final volume of 80 μL. The mixture was incubated at 37°C with shaking (350 rpm) for 45 minutes. The oxidized RNA was purified using ethanol precipitation. Final cleanup steps included the use of Micro Bio-Spin P-6 Gel Columns and the Zymo RNA Clean \u0026amp; Concentrator-5 (Zymo Research, #R1013), with the RNA eluted in 20 μL of nuclease-free water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3’\u003c/strong\u003e\u003cstrong\u003e-DNA Linker Ligation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3’-DNA linker ligation was performed using 20 μL of oxidized RNA, 2 μL of a 3’-DNA linker (10 μM), 2 μL of RNaseOUT inhibitor, 17 μL of 50% PEG 8000, 5 μL of RNA ligase buffer (10×, New England Biolabs, #B0216S), and 4 μL of T4 RNA Ligase 2 truncated KQ (New England Biolabs, #M0373), resulting in a total reaction volume of 50 μL. The reaction mixture was thoroughly mixed, briefly centrifuged, and incubated overnight at 16°C for 18 hours in a thermal cycler. The RNA was subsequently purified using ethanol precipitation and resuspended in 15 μL of nuclease-free water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTBE-Urea Gel\u003c/strong\u003e\u003cstrong\u003e Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGel purification was performed to separate the ligation products from free 3’ DNA linkers. \u003c/p\u003e\n\u003cp\u003eTwo stock solutions were prepared for the TBE-Urea gel. Solution A was made by dissolving 500 g of Urea (Bio-Rad, #1610745) in 500 mL of 40% Acrylamide Solution (Bio-Rad, #1610140) and 100 mL of 10× TBE buffer (Bio-Rad, #1610770). The volume was brought up to 1,000 mL using 1× TBE buffer. Solution B was prepared similarly by dissolving 500 g of Urea in 100 mL of 10× TBE buffer and 500 mL of nuclease-free water, with the final volume adjusted to 1,000 mL using 1× TBE buffer. Both solutions were filtered through a 0.22 μm filter.\u003c/p\u003e\n\u003cp\u003eA 15% TBE-Urea gel was prepared by mixing 37.5 mL of Solution A, 12.5 mL of Solution B, 400 μL of 10% ammonium persulfate (APS; Bio-Rad, #2610700), and 37.5 μL of TEMED (Bio-Rad, #161-0800) to a final volume of 50.4 mL. The mixture was immediately poured between the glass plates of the Bio-Rad Mini-Protean gel casting apparatus, and a comb was inserted. The gel was allowed to polymerize at room temperature for 60 minutes.\u003c/p\u003e\n\u003cp\u003ePrior to electrophoresis, 2× RNA loading dye (New England Biolabs, #B0363S) was added to the RNA sample, which was then incubated at 72°C for 2 minutes and placed on ice for 3 minutes. The samples were loaded, and electrophoresis was performed at 20W for 50 minutes.\u003c/p\u003e\n\u003cp\u003eThe gel was stained with SYBR Gold Nucleic Acid Gel Stain (Invitrogen, #S11494) by incubating it in a 1× TBE staining solution for 15 minutes with gentle shaking. RNA bands were visualized using a Dark Reader Non-UV Transilluminator (Model: DR-46B), and the desired fragment was excised and transferred to a tube. Images of the gel were captured both before and after excision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcrylamide Gel RNA Extraction\u003c/strong\u003e\u003cbr\u003eTo extract RNA product from the acrylamide gel, LoBind tubes were prepared with 1.5 μL GlycoBlue Coprecipitant and 33 μL Sodium Acetate.\u003c/p\u003e\n\u003cp\u003eA hole was pierced in the bottom of a 0.6 mL tube using an 18G needle, holding the needle near the tip and carefully pressing it into the tube’s bottom. The 0.6 mL tube was placed inside a 2 mL tube, and the gel slices were added to the 0.6 mL tube. The assembly was centrifuged at maximum speed for 1 minute at 4°C, allowing the crushed gel to flow into the larger tube.\u003c/p\u003e\n\u003cp\u003eEach tube was supplemented with 300 μL of nuclease-free water, and the mixture was incubated in a ThermoMixer C (Eppendorf) at 70°C for 10 minutes with shaking at 1800 rpm. This incubation was repeated an additional two times. Following incubation, the tubes were briefly centrifuged, and the supernatant was transferred to a Costar SpinX Centrifuge Tube Filter (0.45 μm; #8162). The SpinX tube was centrifuged at 10,000 rpm for 1 minute, and the flow-through was transferred into prepared LoBind tubes containing GlycoBlue and sodium acetate. Ethanol precipitation was performed, and the RNA product was eluted in 22 μL of nuclease-free water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5’\u003c/strong\u003e\u003cstrong\u003e-RNA Linker Ligation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo perform the 5’-RNA linker ligation, 2 µL of 5’-RNA linker (50 µM) was denatured at 72°C for 2 minutes and immediately placed on ice. A ligation reaction mixture was prepared by combining 22 µL of the ligated RNA product, 4 µL of 100% DMSO, 4 µL of T4 RNA Ligase Reaction Buffer (10×, B0216S), 4 µL of ATP (10 mM), and 1 µL of RNaseOUT inhibitor, resulting in a total volume of 35 µL. Next, 35 µL of the prepared ligation mixture was combined with 3 µL of T4 RNA Ligase 1 (New England Biolabs, #M0204S) and 2 µL of the 5’-RNA linker (50 µM), bringing the final reaction volume to 40 µL. The reaction was mixed, briefly centrifuged, and incubated at 25°C for 1 hour. Following the incubation, RNA purification was carried out using the Monarch Spin RNA Cleanup kit (New England Biolabs, #T2030) according to the manufacturer’s instructions, and the RNA product was eluted in 20 µL of nuclease-free water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecDNA Synthesis\u003c/strong\u003e\u003cbr\u003eFor cDNA synthesis, 20 µL of the ligated RNA product was combined with 1 µL of 10 µM Reverse Transcriptase (RT) primer, 2 µL of 10 mM dNTPs, and 2 µL of nuclease-free water in a final volume of 25 µL. The mixture was denatured at 65°C for 5 minutes, immediately placed on ice, and briefly centrifuged. To this, 8 µL of ProtoScript II buffer (5×, New England Biolabs, #B0368S), 1 µL of RNaseOUT inhibitor, 4 µL of 0.1 M DTT (New England Biolabs, #B1034A), and 2 µL of ProtoScript II Reverse Transcriptase (New England Biolabs, #M0368S) were added to bring the final reaction volume to 40 µL. The reaction was incubated at 50°C for 1 hour to synthesize the first strand of cDNA. To hydrolyze the RNA, 4 µL of 1 N Sodium Hydroxide Solution (Fisher Chemical, #SS266-1) was added, and the mixture was incubated at 95°C for 15 minutes. The reaction was neutralized with 4 µL of 1 M Tris–HCl (pH 7.5; Invitrogen, #15567027), and the cDNA was purified using 1.8× AMPure XP Reagent (Beckman Coulter, #A63881) following the manufacturer’s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecDNA Purification Using AMPure XP Reagent\u003c/strong\u003e\u003cbr\u003eThe cDNA was purified by adding 1.8× AMPure XP reagent to the cDNA sample (48 µL cDNA + 86.4 µL beads). The mixture was thoroughly mixed by pipetting up and down 10 times and incubated at room temperature for 10 minutes. The tubes were briefly spun down and placed on a magnetic stand for 5 minutes until the liquid became clear. The supernatant was carefully removed and discarded, and the DNA-bound beads were washed twice with 500 µL of freshly prepared 80% ethanol. After each wash, the beads were incubated on the magnetic stand for 30 seconds, and the supernatant was discarded. Residual ethanol was carefully removed with a pipette, and the beads were air-dried for 5–10 minutes. The dried beads were resuspended in 32 µL of nuclease-free water, incubated at room temperature for 2 minutes, and briefly spun down. The eluate (30 µL), containing the purified cDNA, was carefully transferred to a new tube for subsequent use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLibrary Amplification and Library Quality Control (QC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLibrary amplification was performed using dual-index sequences. The reaction was set up with 30 µL of cDNA, 10 µL of 5× Q5 Reaction Buffer, 1 µL of 10 mM dNTPs, 2.5 µL of 10 µM i7 index, 2.5 µL of 10 µM i5 index, 0.5 µL of Q5 High-Fidelity DNA Polymerase (New England Biolabs, #M0491), and 3.5 µL of nuclease-free water, in a final volume of 50 µL. Thermocycling conditions were as follows: initial denaturation at 98°C for 30 seconds, followed by three cycles of 98°C for 10 seconds, 52°C for 30 seconds, and 72°C for 30 seconds. This was followed by 18 cycles of 98°C for 10 seconds, 62°C for 30 seconds, and 72°C for 30 seconds. The final extension was performed at 72°C for 2 minutes, and the reaction was held at 4°C. The amplified library was purified using ethanol precipitation and P-6 columns, followed by size selection using SPRIselect beads (Beckman Coulter, #B23318) to ensure high-quality libraries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSPRI-Based Size Selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPRI-based size selection was performed using SPRIselect (Beckman Coulter, #B23318) to ensure appropriate library fragment size distribution. For left-side selection, 60 µL of SPRIselect beads (1.2× ratio) was added to a 50 µL amplified library sample. The beads were thoroughly mixed by pipetting up and down 10 times and incubated at room temperature for 1 minute. The reaction tube was then placed on a magnetic stand for 5–10 minutes to allow the beads to settle. Once the liquid clarified, the supernatant was carefully discarded. The beads were washed twice with 500 µL of freshly prepared 85% ethanol. After each wash, the supernatant was removed following a 30-second incubation, and the beads were air-dried for 5–10 minutes to ensure complete ethanol removal. The purified DNA was eluted by resuspending the beads in 52 µL of nuclease-free water. After incubation for 1 minute and magnetic separation, the eluate (50 µL) was carefully collected, leaving the beads behind.\u003c/p\u003e\n\u003cp\u003eFor right-side size selection (0.7×/1.1×), 35 µL of SPRIselect beads (0.7× ratio) was added to the 50 µL library sample and mixed thoroughly. Following incubation and magnetic separation, the supernatant containing the desired DNA fragment was transferred to a new reaction tube, and the discarded beads contained larger fragments. To the supernatant, 55 µL of SPRIselect beads (1.1× ratio) was added to remove smaller fragments. After incubation, magnetic separation, and two ethanol washes, the beads were air-dried, and the DNA was eluted in 22 µL of Resuspension Buffer (Illumina, #15026770). The final eluate containing the size-selected library was carefully transferred to a new LoBind tube and stored at −80°C for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRibOxi-Seq2 Library Preparation and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLibrary size distribution was confirmed using the D1000 ScreenTape and reagents on the 4200 TapeStation system (Agilent, #5067-5582 and #5067-5583). Library concentration was quantified using the Qubit dsDNA BR Assay Kit (Invitrogen, #Q32853) on a Qubit 2.0 fluorometer (Invitrogen). For sequencing, libraries were initially run on an Illumina MiSeq system using the MiSeq Reagent Nano Kit v2 (300 cycles; Illumina, #MS-103-1001) with a loading concentration of 9 pM. For higher throughput sequencing, libraries were diluted to a final concentration of 1050 pM and sequenced on an Illumina NextSeq 2000 system using the NextSeq 2000 P2 Kit (200 cycles; Illumina, #20046812). A sequencing depth of 30 million reads per sample was achieved to ensure sufficient coverage for downstream analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRibOxi-Seq2 analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRibOxi-Seq data were processed following the previously established pipeline (https://github.com/yz201906/RibOxi-seq, https://github.com/xxy103/RibOxi-seq) (2). In brief, sequencing adapters were trimmed using Cutadapt v2.7 (https://github.com/marcelm/cutadapt). Paired-end reads were subsequently merged into single-end reads with PEAR v0.9.11. Unique molecular identifiers (UMIs), consisting of 10 randomized nucleotides, were extracted and appended to the fastq headers using the “move_umi.py” script. Quality-filtered reads (with length longer than 20nt, Q-value over 20) were then mapped to the reference genome GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta using STAR v2.7.1a. The aligned reads were filtered and de-duplicated using UMI-TOOLS v1.1.1. Single-nucleotide 3’-end coverage profiles indicative of Nm sites were calculated using Samtools v1.9 and Bedtools v2.29.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003erRNA, snRNA, and tRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003erRNA and snRNA sequences were retrieved from https://rnacentral.org/, tRNA sequences were downloaded from https://gtrnadb.org/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSnoscan RNA modification prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe annotated snoRNA sequences were downloaded from Human snoDB \u003csup\u003e25\u003c/sup\u003e, the top190 expressed human snoRNAs in 5 human cell lines (HepG2, HEK293T, PC3, A549, MDA-MB-231) were obtained from previous publication \u003csup\u003e62\u003c/sup\u003e. Putative RNA modification sites within these snoRNAs were predicted with snoScan v1.0 \u003csup\u003e61\u003c/sup\u003e, using as reference the Nm regions (± 7nt) identified through RibOxi-Seq2 analysis. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSnoRNA-RNA interactions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe snoRNA-RNA base pairing was computed with snoDB online tools (https://bioinfo-scottgroup.med.usherbrooke.ca/snoDB/sequence_similarity_search/) \u003csup\u003e25\u003c/sup\u003e, predicted interactions were filtered based on alignment scores and thermodynamic plausibility. The RNA-RNA interactions Minimum Folding Energy (MFE) was determined using RNAduplex (ViennaRNA v2.5.1) \u003csup\u003e91\u003c/sup\u003e, which computes the MFE of hybridization between snoRNA and Nm regions (± 7nt) identified from RibOxi-Seq2 analysis. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetagene analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetagene plot summarizing the mRNA features were generated with metaPlotR.\u003c/p\u003e\n\u003cp\u003e(https://github.com/olarerin/metaPlotR)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRiboMeth-Seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH9 hESCs were maintained on Matrigel-coated (Corning, #354277) plates with Essential 8 media (Thermo Fisher, #A1517001). Stem cells were differentiated to neurons for 14 days per the i\u003csup\u003e3\u003c/sup\u003e neuron protocol \u003csup\u003e92\u003c/sup\u003e. hESCs were prepared for differentiation once 70-80% confluent. Differentiated cells were manually removed. Stem cells were released from the plate with Accutase (Sigma-Aldrich, #SCR005) incubation. Media was added to the cell solution and cells were centrifuged. Cells were resuspended in induction media (IM) supplemented with 2µg/ml doxycycline (Dox) (Milipore Sigma, #D9891) and 10µM Rock Inhibitor (Tocris, #1254). IM was made with DMEM/F12 with HEPES (Thermo Fisher, #11330032), N2 supplement (Thermo Fisher, #17502048), MEM Non-essential amino acids (Thermo Fisher, #11140050), and L-Glutamine (Thermo Fisher, #25030081). Cells were plated at 1.5x10\u003csup\u003e5\u003c/sup\u003e cells/well in a 6-well plate. Cells were fed daily with IM and received 2 additional days of Dox treatment. Cells were then replated in IM on poly-D-lysine/laminin-coated 6-well plates (Millipore Sigma P1149, Fisher Scientific, #23-017-015) at 5x10\u003csup\u003e5\u003c/sup\u003e cells/well. The day after plating, the media was fully changed to cortical neuron culture media (CM) which is composed of DMEM/F12 with HEPES and Neurobasal medium (Thermo Fisher, #21103049) supplemented with laminin, BDNF (Thermo Fisher, #450-02-10UG), GDNF (Thermo Fisher, #450-10-10UG), NT3 (Thermo Fisher, #450-03-10) and B27 supplement (Thermo Fisher, #17504044). Subsequently, half media changes with CM were performed on every other day until the 14\u003csup\u003eth\u003c/sup\u003e day of differentiation. Cells were rinsed with PBS and then were collected in TRIzol Reagent (Thermo Fisher, # 15596018). 0.2ml of chloroform per 1ml of TRIzol was added and samples were centrifuged. The aqueous phase was transferred to a new tube and 0.5ml of isopropanol was added. Samples were incubated at -80°C for 10 minutes and then centrifuged to pellet the RNA. The pellet was washed with 75% ethanol and resuspended in nuclease-free water.\u003c/p\u003e\n\u003cp\u003eSamples were prepared in triplicate for RiboMeth-Seq following \u003csup\u003e37\u003c/sup\u003e with some modifications. Briefly, 250ng RNA per sample was subjected to alkaline hydrolysis. Fragmented RNA was then ethanol precipitated and run on a TapeStation to check size distribution. Samples were 3’ end dephosphorylated by incubating for 3 hrs at 37°C with T4 PNK (New England Biolabs, M0201S) and pH6 T4 PNK buffer. Then 5’ end phosphorylation was achieved by adding additional T4 PNK, T4 PNK reaction buffer, and ATP and incubating for 30 minutes. A 3’-DNA linker was ligated to the RNA with T4 RNA ligase 2 truncated KQ (NEB, M0373S). Then a 5’-RNA linker was ligated to the RNA. The linker was denatured and then the ligation reaction was prepared utilizing T4 RNA ligase 1 (NEB, M0204S. cDNA was synthesized using Superscript III (Thermo Fisher, #18080044). The RNA was hydrolyzed and the cDNA purified with AmpureXP beads (Beckman Coulter, A63880). Libraries were amplified with KAPA HiFi DNA polymerase (Roche, #07958927001) and sequenced on Illumina NovaSeq. Adaptor sequences, reverse transcription primers, and PCR primers were applied according to the procedures described above.\u003c/p\u003e\n\u003cp\u003eRiboMeth-Seq Analysis by MethScore was calculated as described \u003csup\u003e35,93\u003c/sup\u003e. The 2 nucleotides flanking each position were used as the flanking region. Briefly, across all positions, the sum of 3’-ends’of profile at the n position and the 5’-ends of profile at the n+1 position was calculated. For each position, the sum of ends was divided by ½ of the sum of the weighted end sum for the flanking positions divided by the sum of the weights. This was subtracted from 1 to give the MethScore. Standard deviation was calculated across 3 replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNm-VAQ Quantitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e500 ng of total RNA from CT2-NGN2 neuron cells was mixed with 50 pmol of the RNA/DNA chimera. The volume was adjusted to 11 µl using 10 µM Tris pH 7.0 buffer. For mRNA-specific targeting, 500 ng of polyA-selected RNA (NEB, #E7490L) was used. Samples were incubated at 95°C for 1 minute and immediately transferred to ice. Subsequently, 5 µl of the annealed RNA/chimera mixture was combined with 1 µl of RNase H enzyme (NEB, #M0297S), 1 µl of 10x RNase H buffer (NEB, #B0297S), and 3 µl of nuclease-free water. The remaining 5 µl of the annealed RNA/chimera mixture was mixed with 1 µl of 10x RNase H buffer and 4 µl of nuclease-free water. The samples were thoroughly mixed by pipetting and incubated at 37°C for 30 minutes.\u003c/p\u003e\n\u003cp\u003eAt this stage, the protocol diverges for highly abundant rRNA and low abundance mRNA. For highly abundant RNAs, the samples were incubated at 90°C for 10 minutes to denature the RNase H enzyme, and then placed on ice. After denaturation, the samples were diluted 1:5. Then, 1 µl of the diluted sample was used for cDNA synthesis with SuperScript III Reverse Transcriptase (Invitrogen, #18080044) and random hexamers. Subsequently, 1 µl of cDNA was used for RT-qPCR with PowerSybr (Applied Biosystems, #4368706).\u003c/p\u003e\n\u003cp\u003eFor low abundance mRNA, the reaction volume after the 30-minute RNase H cleavage step was increased to 30 µl with sterile nuclease-free water, and 30 µl of Phenol-chloroform-isoamyl alcohol mixture (Millipore Sigma, #77617) was added. After vigorous vortexing, the mixture was centrifuged at 12,000 g for 5 minutes. Approximately 20 µl of the upper aqueous phase was transferred to a clean 1.7 mL Eppendorf tube. Cytiva Microspin G-50 columns (Cytiva, #27533001) were prepared by loosening the cap, removing the bottom plug, and placing them into 2 mL collection tubes. Excess buffer was removed by centrifuging at 700 g for 1 minute. The column was then transferred to a clean 1.7 mL Eppendorf tube, and the upper phase from the previous extraction was added to the column. To elute, the tube was centrifuged at 700 g for 2 minutes. 1.5 µl of the eluate was used for cDNA synthesis with SuperScript III and random hexamers. Finally, 1 µl of the cDNA was used for RT-qPCR with PowerSybr. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Gordon G. Carmichael ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRibOxi-Seq2 data for iNs have been deposited at GEO with Bioproject accession number PRJNA1348454. RibOxi-seq data for 293T cells have been deposited at GEO with accession number GSE188194. \u0026nbsp;The scripts for our RibOxi-Seq2 pipeline are available from https://github.com/yz201906/RibOxi-seq. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by NIH grants R35GM118140 to B.R.G., HD099975 and an award from the Foundation for Prader Willi Research to G.G.C., R35GM146883 to J.D.B, F31HD114435 to S.A.A. and R01GM135383 to C.L.H. We thank Sara Olson for helpful suggestions regarding the Illumina sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, G.G.C.; formal analysis, X.Y., Y.L., Y.Z., S.A., B.A.E., and G.G.C; investigation, X.Y., Y.L., Y.Z., S.A.A., B.A.E., and G.G.C; resources, Y.L., Y.Z., S.A. and B.A.E.; writing \u0026ndash; original draft, X.Y. and G.G.C.; writing \u0026ndash; review \u0026amp; editing, all authors; visualization, X.Y., S.A.A., B.E., and G.G.C.; supervision, C.L.H., J.B., B.R.G. and G.G.C.; funding acquisition, C.L.H., J.B., B.R.G. and G.G.C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.R.G. is a co-founder and SAB member for RNAConnect and SAB member of Ascidian Therapeutics.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHelm M, Motorin Y (2017) Detecting RNA modifications in the epitranscriptome: predict and validate. 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Front Genet 11:38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fgene.2020.00038\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fgene.2020.00038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8523796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8523796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe have compared genome-wide patterns of RNA 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders.\u003c/p\u003e","manuscriptTitle":"Genome-wide profiling of RNA 2’-O-methylation in neurons and identification of orphan snoRNA targets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 08:26:12","doi":"10.21203/rs.3.rs-8523796/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"191005e7-66ff-4780-9cb4-8f0841b6fb64","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61241738,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":61241739,"name":"Biological sciences/Molecular biology/Transcriptomics"},{"id":61241740,"name":"Biological sciences/Molecular biology/RNA metabolism/RNA modification"},{"id":61241741,"name":"Biological sciences/Molecular biology/Non-coding RNAs/Small RNAs"}],"tags":[],"updatedAt":"2026-02-19T10:47:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 08:26:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8523796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8523796","identity":"rs-8523796","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}