A novel long intergenic noncoding RNA indispensable for the formation of zygote in pig

A novel long intergenic noncoding RNA indispensable for the formation of zygote in pig | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel long intergenic noncoding RNA indispensable for the formation of zygote in pig Xiaogang Weng, Xu Yang, Yuting zhang, Yuchuan Yang, Jingyu Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4250954/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 Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of mammalian early embryonic development. However, in pig, it is seldom investigated. In this study, lncRNAs were predicted using single-cell RNA-seq data on porcine early embryos from oocyte to early blastocyst. We further identified a novel nuclear long intergenic noncoding RNA (lincRNA), linc-321 , which was specifically and highly expressed in 1-cell embryo, and it was indispensable for the formation of zygote and early embryonic development in pig. Its knockdown by locked nucleic acid (LNA)-siRNA could result in developmental arrest at 1-cell stage with failure of pronuclear envelope fusion (PEF). Mechanistically, linc-321 facilitates the recruitment of SUZ12 to the TSS region of MYT1. And this recruitment is instrumental in establishing H3K27me3 modifications, consequently leading to the repression of MYT1 expression. Moreover, the failure of PEF and developmental arrest induced by loss of linc-321 could be rescued by addition of linc-321 with LNA-siRNA targeting site deletion and MYT1 knockdown. Thus, as functional lincRNA characterized in pig, linc-321 provides the clues for investigating the strictly regulated process of early embryonic development. Biological sciences/Developmental biology/Embryology Biological sciences/Molecular biology/Non-coding RNAs/Long non-coding RNAs long intergenic noncoding RNA early embryonic development formation of zygote epigenetic regulation pig Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Long noncoding RNAs (lncRNAs), a class of longer than 200 nucleotides (nt) and non-translated endogenous cellular transcripts, have been considered as by-products of transcription without functions. However, they have emerged as new and fundamental transcriptional and post-transcriptional regulators acting at multiple levels of gene expression in the nuclear and/or in the cytoplasmic compartments, and generating an intricate network with RNAs, promoters and enhancers, and chromatin-modifier complexes over the past few years 1–4 . Considering the versatility of the molecule to operate in different subcellular compartments, via different modes of action and with different target specificity, the annotation and understanding of lncRNAs in various biological process are desirable. Mammalian early embryonic development, from zygote to blastocyst, is a strictly regulated process 5–7 . Generally, some transcription factors are believed to important for the progression, such as Oct4, which plays an essential role in the development of pluripotent cells in embryo. In addition to the key transcription factors, lncRNAs also participate in a wide variety of the developmental process. For example, promoter-associated noncoding RNAs (pancRNAs), transcribed from bidirectional promoters, have been shown to be indispensable for embryonic development by activation of their partner genes during zygotic genome activation (ZGA) 8 . Moreover, our studies have reported that an endogenous retrovirus (ERV)-associated lncRNA, LincGET , is identified as one of the earliest known lineage regulators to bias cell fate in 2-cell embryo by promoting the nuclear localization of CARM1 and is essential for embryonic development in mice 9,10 . Our latest research has identified the lncRNA named SAWPA as crucial in the early embryonic development of pigs 11 . The above studies emphasize that lncRNAs are essential regulators of early embryonic development in pigs, which mirrors the complexity and specificity of mammalian development. Pig, as an important livestock, is considered as human disease model and potential organ donor for xenotransplantation in regenerative medicine 12 , and shows conserved principles of early development with human 13 . Therefore, it is urgent to comprehensively understand the molecular mechanism on regulation of embryonic development specific to pig. Mammalian early embryonic development shows broad similarities among mammals, but also reveals crucial species differences in the transcriptional and spatio-temporal regulation. In mice, as a key trophoblast (TE) transcription factor, CDX2 can repress the transcription of OCT4 by binding to the fourth conserved region of the distal OCT4 enhancer to form TE cells 14 . However, we have previously demonstrated that CDX2 can not repress the transcription of OCT4 in porcine early embryos, because of lack of the cis -acting regulatory region 15 . To further figure out the specificity in pig, we have performed single-cell RNA-seq (scRNA-seq) on porcine early embryos, from 1-cell to early blastocyst (EB), and revealed the molecular features of the first lineage specification, the emergence of pluripotency, the transition of epigenetic landscapes and the dynamics of X-chromosome dosage compensation during the early development 16 . In the study, we profiled the transcriptome of lncRNAs during the early embryonic development in pig using the scRNA-seq data. And we identified a novel nuclear lincRNA, linc-321 , which is essential for the formation of zygote in pig. 2 Materials and Methods 2.1 Porcine embryo culture and collection All experiments were performed according to the guide-lines of The State Key Laboratory Animal Care and Use Committee. The procedure for porcine IVF has been described previously. Briefly, freshly ejaculated sperm-rich fractions were collected from fertile boars. Following short incubation at 39°C, semen was resuspended and washed three times in DPBS supplemented with 0.1% (w/v) BSA via centrifugation at 1500 g for 4 min. Spermatozoa concentrations were measured using a hemocytometer, and the proportion of motile sperm determined. Next, spermatozoa were diluted with modified Tris-buffered medium (mTBM) to an optimal concentration. Cumulus-free oocytes were washed three times in mTBM. Approximately 30 oocytes were inseminated in 50 ml mTBM at a final sperm concentration of 3 × 10 5 /ml for 5 h. Embryos were cultured in porcine zygote medium-3 (PZM-3) at 39°C in 5% CO 2 in air. Embryos were collected after IVF at the following time points: 1-cell stage (24 hours), 2-cell stage (40–45 hours), 4-cell stage (65–72 hours), 8-cell stage (84–90 hours), morula stage (108–115 hours) and blastocyst stage (156–160 hours). Besides, the oocytes were collected at 42 h in vitro maturation. For qPCR, about 50 embryos of each stage were used. For α -Amanitin treatment, different concentrations (1µg/ml, 5µg/ml and 10µg/ml) of α -Amanitin (HY-19610, Med Chem Express) was added into culture medium at 6 hours after oocyte activation. To knockdown of linc-321 , we injected about 10 pl 10 µM LNA-siRNA targeting on linc-321 into per MII oocyte, and then performed IVF to obtain embryos. The sequences of siRNAs are shown in Table S10 . For rescue experiment, about 10 pl mixture of 200nM mutant linc-321 from in vitro transcription was also injected into per MII oocyte. Embryo development was then observed every 24 hours. 2.2 TRIM-Away assay pSMPP-mCherry-hTRIM21 vector was purchased from Addgene ( https://www.addgene.org/104972/ ). mCherry-hTRIM21 was cloned into pCMV6-XL6 expression vector ( https://www.addgene.org/vector-database/5211/ ) using EcoRI and XmaI restriction sites. To make in vitro transcribed mCherry-hTRIM21 mRNA, the template DNA was linearized by AgeIHF digestion. mRNA was generated by in vitro transcription using mMESSAGE mMACHINE T7 Transcription kit (#AM1344; Thermo Fisher Scientific) and purified using RNeasy MinElute Cleanup Kit (#74204; Qiagen). mRNA was aliquoted at a concentration of 2 mg/ml and stored at -80°C. CDC2 antibody (Abcam, ab18) was used. About 10 pl the mCherry-TRIM21 mRNA and CDC2 antibody complex was injected into MII oocyte, and IVF was preformed after injection. 2.3 RNA-seq data quality control, processing and analysis Sequencing reads obtained from our previous single cell RNA-seq were assessed with the fastX-toolkit (version 0.0.13) to remove short (-l 20) and low quality (-q 20) reads, followed by trimming of the adaptor sequence. The qualified reads were aligned with the Tophat 2 17 into the porcine reference genome (Suscrofa 10.2.87) by default parameters. The reads on the extracted alignment were constructed with Cufflinks to construct transcripts. The transcripts of all samples were combined and reconstructed into a large transcript file (merged.gtf) using Cuffmerge 18 . Then the expression of genes in each sample were quantified to FPKM by Cuffnorm 18 . For differential expression, we first counted the overlap of reads with genes by htseq-count 19 with the parameter “-m union”. Next, we compared the two groups using default parameters in R package: DESeq2. A gene was considered significant if the Benjamini and Hochberg–adjusted Pvalue (Padj) was less than 5% and the fold-change was greater than 2. And we used Short Time-series Expression Miner (v1.3.11) for analysis of gene expression trends. 2.4 LincRNA detection pipeline. To identify lincRNAs in pig, we designed an analysis pipeline to minimize false positives and maximize the number of lincRNA transcripts, including the following five steps: ( 1 ) we used Cuffcompare to compare our merged transcriptome with annotation in Ensembl databases, and removed potential known transcripts; ( 2 ) filter transcripts that are shorter than 200 nt; ( 3 ) select transcripts that are more than 2 exon; ( 4 ) keep only transcripts that are located at least 500 bp away from any protein-coding genes or house-keeping ncRNAs genes annotated in the Ensembl Sus scrofa10.2 gene set (GTF); ( 5 ) filter putative lincRNA transcripts by coding potential using the PLEK 20 , CPC2 21 and CPAT 22 softwares, which are independent of known annotations and have been proved the best effective lncRNA identification. 2.5 Unsupervised hierarchical clustering Using all expressed genes as input was conducted on all filtered cells by normalized read counts in log 2 scale. The distance method was Euclidean, and the cluster method was ward. D2. 2.6 Immunofluorescence (IF) analysis IF analysis of porcine embryos was conducted as previously described 8 . After removal of the zona pellucida with acidic MAN solution, embryos were fixed in 4% PFA for 30 min at room temperature. After three washes for 5 min each in washing solution (0.1% Tween-20, 0.01% Triton X-100 in 1× PBS), embryos were permeabilized in normal permeabilizing solution (1% Triton X-100 in 1×PBS) for 20 min at room temperature. Embryos were then blocked in blocking solution (1% BSA in 1× PBS) for 1 h at room temperature after three washes for 5 min each in washing solution, followed by incubation with primary antibody diluted with blocking solution overnight at 4°C. After three washes for 5 min each in washing solution, embryos were incubated with secondary antibody diluted with washing solution for 1 h at room temperature. After three washes in washing solution, nuclei were stained with Hoechst 33324 for 7 min. Embryos were then mounted on glass slides after three washes. Antibodies in this research: β-Tubulin (Covance, MRB-435p), Lamin (Abcam, ab26300), CDC2 (Abcam, ab18). 2.7 Strand-specific RT–PCR (SSRT-PCR) SSRT-PCR was performed using Reverse Transcriptase M-MLV (RNase Hˉ) Kit (TaKaRa). Briefly, mix 1ng ~ 1µg template RNA and 1µL specific primer (F or R) in the tube, and add RNase free H 2 O to 6µL. Holding the template RNA/primer mixture at 70℃ for 10 min, then rapidly cooled on ice for 2 min. Add 2 µL 5×M-MLV Buffer, 0.5µL dNTP Mixture (10 mM each), 0.25µL RNase Inhibitor (40 U/µL), 1µL RTase M-MLV (RNase Hˉ) (200 U/µL) and 0.25µL RNase free H 2 O in above tube. After mix the mixture, holding the mixture at 42℃ for 60 min, 70℃ for 15 min. Then perform PCR reaction use above reaction products. 2.8 Western blot (WB) The protein retrieved from 200 embryos digested with Pierce IP lysis buffer (10 µl/lane) was mixed with 30 µl sample buffer (10 ml; 1.25 ml 0.5 M-pH 6.8-Tris–HCl, 2.5 ml glycerin, 2 ml 10% SDS, 200 µl 0.5% bromophenol blue, 3.55 ml H2O, and 0.5 ml b-mercaptothion) and incubated for 5 min in boiling water. The samples were separated on SDS–PAGE with a 5% stacking gel (10 ml; 5.7 ml ddH2O, 2.5 ml 1.5 M pH 6.8 Tris–HCl, 1.7 ml 30% acrylamide (acryl: bis acryl = 29:1), 100 µl 10% SDS, 50 µl 10% ammonium persulfate, and 10 µl TEMED) and a 10% separating gel (10 ml; 4.1 ml ddH2O, 2.5 ml 1.5 M pH 8.8 Tris–HCl, 3.3 ml 30% acrylamide (acryl: bis acryl = 29:1), 100 µl 10% SDS, 50 µl 10% ammonium persulfate, and 5 µl TEMED) at 120 V for 1.5 h and then electrophoretic ally transferred onto a nitrocellulose membrane at 300 mA for 1 h. Membranes were blocked in TBST buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4) containing 3% BSA, for 1 h at RT and then incubated with primary antibody, diluted in TBST containing 1% BSA, overnight at 4°C. After three washes for 10 min each in TBST, the membrane was incubated for 1 h at RT with the secondary antibody diluted in TBST. After three washes for 10 min each, the signals were detected using ECL and films. Antibody used: MYT1(Santa, sc-398299), CDC2-p34(Santa, sc-136014). 2.9 Real-time RT-PCR (qPCR) analysis. Total RNA was extracted using the PureLink™ Micro-to-Midi System (Invitrogen) according to the manufacturer’s instructions, and reverse transcription was used to generate cDNAs using the PrimeScript™ RT Reagent kit (TaKaRa). qPCR was performed using SYBR Premix Ex Taq™ (TaKaRa) and the 7500 Real-Time PCR System. The reaction parameters were 95°C for 30 s followed by 40 two-step cycles of 95°C for 5 s and 60°C for 34 s. Ct values were calculated using Sequence Detection System software, and the amount of target sequence normalized to the reference sequence was alculated as 2 −ΔΔCt . All primers and probes were designed using Primer Premier 5 (Table S10 ). 2.10 Subcellular localization analysis RNA fractionation Cytoplasmic & Nuclear RNA Purification Kit (Norgen Cat. 21000) was used to prepare the nucleus and cytoplasm separately, according to manufacturers’ protocols. In brief, 200 embryos were collected to an RNase-free tube and centrifuge at 2,000 RPM for 10 minutes. Add 200ul of ice-cold Lysis Buffer J to the tube. Lyse embryos by vortexing for 15 seconds. Spin lysate for 3 minutes at 16,000 RPM in a benchtop centrifuge. Cytoplasmic RNA is in the supernatant, and nuclear RNA is in the deposition. Collected the supernatant and deposition separately, and add 200ul of Buffer SK and 200ul of 100% ethanol to both RNA fractions to binding RNA to column. Then, wash the column with 400ul Wash Solution A three times. After that, place the column into a fresh Elution tube, and add 50ul of Elution Buffer E to the column. Centrifuge for 2 minutes at 2,000 RPM, followed by 1 minute at 14,000 RPM to collected cytoplasmic RNA and nuclear RNA separately. The following steps are as for Real-time RT-PCR (qPCR) analysis. RNA-FISH were also performed to confirm the subcellular localization of linc-321 . The probes were labeled by in vitro transcription using DIG RNA Labeling Kit (SP6/T7) (Roche, #11175025910). After removal of the zona pellucida with acidic MAN solution, porcine embryos were incubated in 1×PBS containing 6 mg/ml BSA for 15 min. Then, embryos were transferred on coverslips coated with Denhardt’s solution (Sigma, #30915-5ML) and dried for 30 min at RT. Embryos were fixed in 3% paraformaldehyde (PFA) for 12 min followed by permeabilization in RNA-FISH permeabilizing solution (0.5% Triton X-100, 10 mM Vanadyl ribonucleotide complex (Sigma, #94742-1ML), in 1× PBS) for 6 min on ice. After two washes in 70% EtOH for 5 min each, dehydration was performed in 80%, 95%, twice 100% EtOH, each for 5 min at RT, and the slides were dried for 5 min. The embryos were hybridized in hybridization solution (50% formamide (Sigma), 2× SSC, 10% dextran sulfate (Sigma), 10 mM VRC, 2 mg/ml BSA (Sigma)) containing 0.1 nM DIG-labeled RNA probes at 37°C overnight. After three washes for 5 min each in hybridization washing solution (50% formamide, 2×SSC) at 42°C and four washes for 5 min each in PBT (1% Tween-20, in 1× PBS), we blocked the embryos in blocking solution (10% sheep serum, 0.05% BSA, in 1× PBS) for 1 h at RT followed by incubation in antibody hybridization solution (2% sheep serum, 0.05% BSA, anti-DIG antibody (1:200), in 1× PBS) for 2–3 h at RT. After four washes for 5 min each in PBT, embryos were stained with Hoechst 33342 for 7 min. Then, embryos were mounted on glass slides after three washes. 2.11 Analysis of gene differential expression We compared the two groups using default parameters in R package: DESeq2. A gene was considered significant if the Benjamini and Hochberg–adjusted Pvalue (Padj) was less than 5% and the fold-change was greater than 2. And we used Short Time-series Expression Miner (v1.3.11) for analysis of gene expression trends. 2.12 ELISA Porcine myelin transcription Factor 1 (MYT1) ELISA kit (RJ28474, RENJIEBIO) was used. Capture antibody is pre-coated on the micro titer plate. Firstly, adding standard and samples to wells. Anaylte present was bound by the immobilized antibody. Unbound materials were removed. Secondly, biotin conjugated to detection antibody was added and binded to analyte absorbed on the plate. Unbound ones were washed away. Then, avidin conjugated to HRP was added and binded to biotin absorbed on the plate. Unbound ones were washed away. Finally, add luminal substrate and incubate 10 minutes at 37℃, and read relative light unit value immediately. 2.13 RNA-binding protein immunoprecipitation (RIP) Magnetic Beads Protein G were coated with 5 µg of primary antibody in RIP wash buffer (50 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 1 mM MgCl 2 , and 0.05% NP-40) containing 5% BSA 3h with rotation at 4℃. Then, we collected 2,000 embryos, and added 100 µL of RIP lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM PMSF/cocktail) and 10 µL of RNase inhibitor (Ambion, AM2694) followed by incubation on ice for 10 min. Next, we centrifuged the RIP lysate at 14,000 rpm for 10 min at 4℃, removed 100 µL of the supernatant and added this to 900 µL of beads-antibody complex in RIP Immunoprecipitation Buffer (860 µL RIP wash buffer, 35 µL 0.5 M EDTA, and 5 µL RNase inhibitor), and incubated this with rotation overnight at 4℃. The residual 10 mL of the supernatant of RIP lysate was treated as input. After washing, treated with proteinase K at 55℃ for 30 min with shaking to digest the protein, followed by RNA extraction from the supernatant; qPCR was then performed to detect linc-321 . Antibody used: SUZ12(Abcam, ab12073); IgG (Millipore, CS200621) 2.14 Chromatin Immunoprecipitation (ChIP) assay We collected about 2,000 1-cell embryos and added 100 µl ChIP buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM PMSF/cocktail) and 5 µl RNase inhibitor (Ambion, #AM2694) followed by incubation on ice for 10 min. We then harvested 10 µl of lysates used as inputs. The remaining lysates were divided into two parts and incubated with anti-H3K27me3 (Millipore, 07-499) or anti-H3K9me3 (Abcam, ab8898) antibody overnight at 4°C. Next, 10 µl of protein A agarose beads (Novex, #15918014) was added and the mixture was rotated for 4 h at 4°C, followed by microcentrifugation at 900 g for 5 min at 4°C. The beads were washed three times with ChIP buffer for 5 min each. qPCR was then performed. The sequences of primers to detect H3K27me3 level at MYT1 (± 1.5kb) and GAPDH (± 1.5kb) promoter region were shown in Table S10 . 2.15 EDU staining EDU (10 µM, final concentration) was added to the cultured porcine embryos injected with control-LNA or LNA targeting linc-321 before IVF. After removal of the zona pellucida with acidic MAN solution, embryos were washed two times for 5 min each in washing solution at IVF 12 h. Embryos were then fixed with 3% PFA in 1× PBS for 30 min, followed by permeabilization with normal permeabilizing solution. Incorporated EU was detected using the click-iT RNA Alexa Fluor 488 Imaging Kit (Invitrogen, #C10329). 2.16 RNA-Protein Interaction Prediction (RPISeq) RNA-Protein Interaction Prediction was performed by online software ( http://pridb.gdcb.iastate.edu/RPISeq/index.html ). 2.17 Statistical analysis Statistical analysis was performed using SPSS 19.0 for MicroSoft™ Windows. Data are presented as means ± s.d. The Least Significant Difference method was employed for multiple comparisons. Data were considered statistically significant at P < 0.05. 3 Results 3.1 Transcriptomic profile of lincRNAs during early embryonic development in pig Some previous studies have annotated lncRNAs in pig. However, these low-depth datasets are difficult to de novo annotate lncRNAs accurately due to their low expressions. To comprehensively understand the lncRNA profile during early embryonic development in pig, we annotated lncRNAs using our previous scRNA-seq data. More than 90 single cells from 1-cell embryo to EB were employed to get sequencing libraries. For each sample, we sequenced an average of 20 Gb data 16 , and 15 Gb of clean data were generated, which covered an average of 36 million reads. To fully and accurately annotate lncRNAs in porcine early embryos, we combined the data from the same stage samples. We identified 532,564 transcripts in total, and more than 80% known protein-coding genes was confirmed 16 . 41,235 novel lncRNA transcripts from 17,636 loci were found (Fig. 1 a), and most of the lncRNAs (68%) were long intergenic noncoding RNAs (lincRNAs; Fig. 1 b). Consistent with lincRNAs identified in mouse and human, the transcript length and the expression level of the lincRNAs in pig were shorter and lower than those of coding genes, and also the lincRNAs contained less number of exons (Fig. 1 c). Unsupervised hierarchical clustering of the lincRNAs (UHC; 28,040 transcripts) grouped the cells according to their developmental stage (Fig. 1 d; Table S1 ). We found MII oocyte, 1-cell and 2-cell embryos were separated from the embryos at the later stages from 4-cell embryo to EB. In addition, 4-cell embryo was divided from the cluster of 8-cell embryo, morula and EB, which may be related to zygote gene activation (ZGA) occurring at the 4-cell to 8-cell stages in pig. And, the principal-component analysis (PCA) of the lincRNAs exhibited the similar clustering pattern (Fig. 1 e). The results show that lincRNAs provide a clear visualization of the developmental progress of porcine early embryos. 3.2 Knockdown of linc-321 result in developmental arrest at 1-cell stage. Lots of lincRNAs were found to be highly expressed in 1-cell embryo (Table S1 ) and inhibiting the transcription in 1-cell embryo by α-Amanitin treatment resulted in majority of embryos arresting at the 1-cell stage (Table S2 ), suggesting the transcripts highly expressed in 1-cell embryos, such as the lincRNAs, may be functional during early embryonic development. To identify the functional lincRNAs, we focused on the top 20 highly expressed lincRNAs in 1-cell embryo (Fig. 2 a; Table S3 ). By injection of LNA-siRNA into oocytes, then used to perform in vitro fertilization (IVF), to knockdown the expression of the lincRNAs, TCONS_00171321 (termed linc-321 ) and TCONS_00192115 (termed linc-115 ) were found to significantly reduce the ratio of IVF embryos developing to blastocyst in vitro , and the knockdown of linc-321 also caused the lower ratio to 2-cell stage, indicating the loss of linc-321 caused the developmental arrest at the 1-cell stage (Fig. 2 b; Table S4 ). And, by NCBI ORF Finder ( https://www.ncbi.nlm.nih.gov/orffinder/ ) analysis, only several mini-ORFs were recognized in linc-321 (Fig. S1 ), confirming its noncoding nature. Thus, in the study, we wanted to show the molecular mechanism of linc-321 on early embryonic development in pig. We first screened all the transcripts from the locus expressing linc-321 . There were 42 transcripts from the locus, and their expression patterns were different during the early development of pig, but only linc-321 was highly expressed in 1-cell embryo (Fig. 2 c; Table S5 ), suggesting in 1-cell embryo, linc-321 is the most representative and functionally active transcript. The sequences of transcripts from the locus were overlapped (Fig. S2 ). To confirm that the developmental arrest at 1-cell stage is contributed by linc321 deletion specifically, we designed a linc-321 -specific LNA-siRNA (321si-2), which targets the second exon of linc-321 and can not target the other transcripts from the locus (Fig. 2 d; Fig. S2 ). We screened the genome-wide profiling of off-targets for 321si-2 by Blastn, and, in addition to the locus expressing linc-321 , another targeting site was found at chr. 3 (Table S6 ), but there was no transcript derived from the locus (Fig. S3 ), confirming the negative off-targets of 321si-2. The high efficiency of linc-321 knockdown by the two siRNAs was verified (Fig. S4 ), and the consistent result was observed (Fig. 2 e; Table S4 ), revealing the functional role of linc-321 on the transition of 1-cell to 2-cell embryos. 2407 bp of the full length of linc-321 with eleven exons, from 89,805,061 to 89,995,64 at chr. 7, was obtained by 5’ and 3’ RACE (Fig. 2 d; Table S7 ). To further avoid the off-target effects by the siRNAs, we deleted the targeting site of 321si-2 in linc-321 and added the siRNA targeting site deleted one (sd linc-321 ) back after linc-321 knockdown. As expected, the developmental deficiency was rescued by the addback experiment (Fig. 2 f; Table S8 ). To determine the transcription direction of linc-321 , we performed strand-specific RT–PCR (SSRT–PCR) analysis and found that linc-321 was transcribed from the bottom strand (Fig. 2 g). RT-PCR (Fig. 2 h) and qPCR (Fig. 2 i) were used to check the expression pattern of linc-321 during the early embryonic development, and the high expression level at the 1-cell stage was verified. We identified a novel lincRNA, linc-321 , and its knockdown resulted in the developmental arrest at 1-cell stage. 3.3 Knockdown of linc-321 results in failure of male and female pronuclear envelope fusion (PEF). It has been demonstrated that the developmental arrest at 1-cell stage may be due to the abnormalities of spindle assembly 23 . To demonstrate the mechanism of the developmental arrest by knockdown of linc-321 , we first checked if spindle assembly was affected by linc-321 knockdown in 1-cell embryo at 18h post-IVF by IF analysis of β-Tubulin (Fig. 3 a). Normal spindle was observed in control (21/60) and negative control groups (19/57), but in linc-321 knockdown ( linc-321 KD) group, rare formation of normal spindle was observed (2/53). In addition to that, we observed most of 1-cell embryo with two pronuclei (51/53) in linc-321 KD group (Fig. 3 a). To further confirm the phenomenon, we tested the nuclear morphology at 18h and 24h post-IVF by DNA staining (Fig. 3 b and c) and found the embryos in linc-321 KD group were abnormally arrested at the two pronuclei phase, indicating linc-321 works at an earlier time point. Then, we examined whether linc-321 KD could cause the delay of pronuclear formation. DNA staining on 1-cell embryo at 8h post-IVF was performed, and no significant difference on pronuclear formation among three groups was observed (Fig. 3 d), suggesting linc-321 has no effect on pronuclear formation. Furthermore, we checked whether linc-321 functions on DNA replication. By EDU dying on 1-cell embryo at 10h post-IVF, we found that linc-321 KD did not affect DNA replication (Fig. 3 e). Given that the pronuclei could sustain to 24h post-IVF in linc-321 KD group, we further detected the role of linc-321 on male and female PEF. IF analysis of LAMIN, the main component of nuclear membrane 24 , was performed. In 1-cell embryo at 8h post-IVF, the nuclear envelope could be observed in both control and linc-321 KD groups. However, at 12h, in control group, PEF had happened, and the zygote was forming, but contrastly, most of linc-321 KD embryos still maintained two pronuclei and could not form zygote (Fig. 3 f). To further confirm the function of linc-321 on PEF, we checked its expression levels every 2 hs from 2 h to 16 h post-IVF in 1-cell embryo and found linc-321 was highly expressed specifically at 10 h, just before PEF (Fig. 3 g). The results show linc-321 is essential for the formation of zygote by facilitating PEF at 1-cell stage. 3.4 Linc-321 regulates PEF by inhibiting the expression of MYT1 . LncRNA plays important role on regulation of gene expression by positive and negative ways 25,26 . To demonstrate the mechanism of linc-321 , we tried to screen the genes may be regulated by linc-321 and related to cell cycle. The expression of linc-321 was significantly high in 1-cell embryo, comparing to oocyte and 2-cell embryo. So, we preformed differential expression analysis between 1-cell embryo and oocyte, and 1-cell and 2-cell embryos, to screen the genes with specifically low or high expression level (fold change > = 5) in 1-cell embryo, which were considered as candidates regulated by linc-321 . Among the genes, we found a cell cycle-related gene, MYT1, was significantly low in 1-cell embryo, comparing to oocyte and 2-cell embryo (Fig. 4 a). Previous studies have shown that MYT1 can phosphorylate CDC2, a major component of Mature Promoting Factor (MPF), to decrease the activity of MPF 27,28 . MPF can promote LAMIN phosphorylation to break down the nuclear envelope, and partial fusion of the male and female pronuclear envelope is the prerequisite for PEF 29 . So, we first check the role of MPF on PEF. We detected the activity dynamics of MPF during the process of PEF by IF detecting the protein level of CDC2. The result showed that MPF activity was in a low level just after fertilization at 4 h post-IVF and significantly enhanced from 10 h to 12 h post-IVF, the time that PEF occur (Fig. 4 b, Fig. S5 ). And we observed that the knockdown of CDC2 protein by TRIM-Away resulted in the failure of PEF (Fig. 4 c). The results suggest that MPF may be essential for PEF. So, we checked if linc-321 can regulate the activity of MPF by regulation of MYT1 expression to perform PEF in porcine 1-cell embryo. As expected, the expression of MYT1 was significantly increased by linc-321 KD (Fig. 4 d). Furthermore, the protein level of MYT1 was increased, and phosphorylated CDC2 (p-CDC2), was upregulated (Fig. 4 e and f). Also, the activity of MYT1 was increased by linc-321 KD detected by ELISA (Fig. 4 g). Then, we examined if the knockdown of MYT1 could rescue the abnormalities by linc-321 KD. We observed the injection of MYT1 antibody (MYT1-Ab) could efficiently inhibit the increasing of MYT1 and p-CDC2 by linc-321 KD (Fig. 4 e and f). PEF occurred normally in linc-321 and MYT1-knockdowned 1-cell embryos at 12h post-IVF (Fig. 4 h), and the in vitro development of linc-321 -deficient embryos was rescued by MYT1-Ab and MYT1 siRNA (si- MYT1 ) injection (Fig. 4 h; Fig. S7 ; Table S9 ). We also found the deficiency of MYT1 by MYT1-Ab injection had no effect on the in vitro development, indicating other nuclear kinases, such as WEE1, may compensate for the loss of MYT1 to inhibit CDC2 by phosphorylating (Fig. 4 i). Furthermore, to confirm that the developmental arrest in linc-321 -deficient embryos was led by the failure of PEF, 321si-2 was injected into 1-cell embryos at 14h post-IVF. As expected, the in vitro development was not affected by the knockdown (Fig. 4 i). The result demonstrated linc-321 regulates male and female PEF by inhibiting the expression of MYT1 . 3.5 Linc-321 establishes H3K27me3 to repress expression of MYT1 by recruiting PRC2 complex. To figure out how linc-321 inhibits the expression of MYT1 , we first investigated the subcellular localization of linc-321 . qPCR (Fig. 5 a) and RNA-FISH (Fig. 5 b and c) showed linc-321 was mainly in the nucleus, indicating the inhibition of MYT1 by linc-321 may be at the transcriptional level. It has been demonstrated lncRNA could regulate gene expression by establishment of epigenetic modification 3,30 , and H3K27me3 and H3K9me3 are the main inhibitory modifications. Polycomb Repressive Complex 2 (PRC2) is comprised of EZH2, SUZ12, and EED, and responsible for establishment of H3K27me3 30 . SETDB1 and SETDB2 are responsible for establishment of H3K9me3 31 . To test the interaction of the above proteins with linc-321 , RNA-protein interaction was predicted by RPISeq, and we found that the interaction of linc-321 with SUZ12 was positive (PIP-SVM = 0.93). Then, RIP was performed using SUZ12 antibody to evaluate the interaction. We found linc-321 could be enriched by SUZ12, comparing to IgG (Fig. 5 e). Thus, we checked the effect of linc-321 knockdown on the alteration of H3K27me3 at MYT1 promoter region (TSS ± 1.5kb) by ChIP-PCR. The GAPDH promoter region was also detected as control. The significant loss of H3K27me3 was observed in linc-321 -deficient embryos, and the sd linc-321 could rescued the loss (Fig. 5 d), suggesting linc-321 may establish H3K27me3 to repress the expression of MYT1 . LncRNAs are known to interact with DNA by direct base pairing to assemble protein complex on chromatin at specific targeting sites 3,32 . We found a 20nt sequence of linc-321 was complementary with the sequence at -6,449 to -6,430 of MYT1 TSS (Fig. 5 f), indicating the sequence might mediate the lncRNA-DNA interaction. To confirm the interaction, we addback the mutant linc-321 with double deletion of the siRNA targeting site and the sequence predicted to perform the interaction (dd linc-321 ) when 321si-2 injection. Unlike sd linc-321 , we found dd linc-321 could not rescue the developmental deficiency (Fig. 2 f; Table S8 ), and ChIP-qPCR show that the enrichment of H3K27me3 signal at MYT1 TSS was also in a significant low level (Fig. 5 d), indicating the lncRNA-DNA interaction may mediate by the complementary sequences. The results indicate linc-321 could recruit PRC2 and establish H3K27me3 to repress expression of MYT1 at the transcriptional level. 4 Discussion In the expanded view of both genomic and transcriptomic analysis, thousands of lncRNAs have been identified in mouse and human 33–35 . As the majority of loci transcribed into lncRNA display poor conservation 33 , the characterization of lncRNAs in other species is needed. In the study, taking advantage of high-throughput scRNA-seq, we performed comprehensive analysis of lincRNAs during early embryonic development in pig. More importantly, we found linc-321 , a novel nuclear lincRNA highly expressed in 1-cell embryo specifically, could facilitate pronuclear envelope fusion by establishing H3K27me3 to repress expression of MYT1 , and was shown to be indispensable for formation of zygote in pig (Fig. 6 ). The prediction of lncRNAs in pig is seldom concerned. In our previous study, we have predicted lincRNAs in the early embryos, using the publicly available low input RNA-seq data, and predicted some lincRNAs might play important role on the early developmental process 36 . However, the datasets are collected from different sequencing platforms, and the sequencing depth and biological repeats are inadequate, so the prediction may be not accurate. To more systematically annotate lincRNA in porcine early embryos, in the study, we employed our scRNA-seq data on more than 90 single cells from 1-cell embryo to EB, and the sequencing depth is more than 15G, which will be beneficial to the annotation. To further accurate the annotation, three softwares were combined to predict the coding potential of the transcripts. And, comparing to our previous study 36 , more lincRNAs were identified, which establishes foundation to reveal the functional role of lincRNAs on early embryonic development in pig. Although many lincRNAs have been indicated to play important roles on mammalian preimplantation development 26,35,36 , only a few have been functionally and mechanistically characterized. In the study, we identified a novel nuclear lincRNA, linc-321 , in pig. The full length of linc-321 is 2407bp with eleven exons, from 89,805,061 to 89,995,64 at chr. 7. By careful analysis by qPCR, we found it specifically and transiently expressed at 10h post-IVF. The collection of the 1-cell embryos for scRNA-seq were at 20h post-natural mating. It has been reported that sperm may take 10–12 hs to enter into the oocyte 37,38 . Thus, the expression patterns of linc-321 checked by scRNA-seq and qPCR are consistent. In general, lncRNAs display low expression levels 32,39 , but the functional ones with defining roles show spatio-temporal expressions in specific tissues and cells, such as LincGET , which transiently high expressed in the nucleus of one cell of the late 2-cell mouse embryo, to bias the cell fate to ICM. There are 42 transcripts from the locus expressing linc-321 , and their expression patterns are different during the early development of pig. However, given that the development of the deficient embryos is inhibited at the 1-cell stage, we consider linc-321 as the functional transcript, because it is the only transcript highly expressed in 1-cell embryo. Thus, in 1-cell embryo, we believe linc-321 is the most representative and functionally active transcript, and the specific expression pattern of linc-321 indicates its important function on specific events. Embryogenesis begins from sperm entering oocyte. The male and female chromosomes are first decondensed and the pronuclei is formed. Then, DNA replication on male and female genomes occurs in the pronuclei, respectively. After that, the male and female pronuclear envelope are partially fused to form the zygote, and the zygote enters mitotic phase. The cleavage of embryo is initiated 37,38 . The formation of zygote happens about at 12h post-IVF in pig 38,40 . linc-321 is just highly expressed before the time point, and we found linc-321 -deficient embryos could not form the zygote, due to abnormal male and female pronuclear envelope fusion. And we oberved linc-321 was mainly in the nucleus. After 12h post-IVF, the nuclear envelop of zygote begins to breakdown, and linc-321 may spread into cytoplasm and be degraded by some nuclease. In mammalian embryos, ZGA occurs as early as the 1–4 cell stage. In mice, the initial ZGA that occurs between S phase of the 1-cell embryo and G1 of the 2-cell embryo is designated as minor ZGA to discriminate it from the burst of transcription that occurs during the mid-to-late 2-cell stage, which is designated as major ZGA 41 . The transcription of minor ZGA is relatively promiscuous, low-level, and genome-wide, and a large number of lncRNAs are produced at that time 42,43 . In the study, the 1-cell embryo of pig analyzed were just around the S phase at 10h post-fertilization, indicating the lincRNAs we identified highly expressed at that time may be derived from minor ZGA, and linc-321 is one of them. In mice, it has been demonstrated that the transient inhibition of minor ZGA results in compromised development beyond the 2-cell stage 43 , and we also found the inhibition of the transcription in 1-cell embryo of pig led to the developmental arrest, suggesting the important role of the lincRNAs derived from minor ZGA on early embryonic development. Generally, during oocyte meiosis and early embryonic mitosis, phosphorylation mediated by CDC2, a main component of MPF, is essential for nuclear envelope breakdown, and its phosphorylation resulting in the low activity of MPF can sustain nuclear envelope 44,45 . In oocyte, high activity of MPF is accumulated 45 . After sperm entering into oocyte, the activity of MPF is reduced, and the formation of male and female pronuclei could occur normally, and then the pronuclear envelope are partially fused to form the zygote 45 . However, with the low activity of MPF, the male and female pronuclei could not be breakdown, and PEF could not happen. Thus, the molecular mechanism on PEF with low MPF activity is not clear. In the study, we suggest the increased activity of MPF is critical for the partial breakdown of nuclear envelop of male and female pronuclei, and we demonstrate the transiently high expression of linc-321 to repress the expression of MYT1, a Wee1-related kinase, which can phosphorylate CDC2 on Thr 14 and Tyr 15 to inhibit activity of MPF 28 , can increase the activity of MPF shortly to promote PEF to form the zygote. The loss of linc-321 leads to high expression of MYT1 and the activity of MPF can not be recovered, so the male and female nuclear envelope maintains. LincRNAs can regulate gene expression by many ways depending on their cellular localization 46 . Cytoplasmic lincRNAs can act as competing endogenous RNAs (ceRNAs) to regulate other RNA transcripts by sponging shared microRNAs 4,47 . And, many nuclear lincRNAs can interact with chromatin-associated proteins, acting as protein scaffolding 3,48 . Linc-321 is mainly in the nucleus, so we suppose it may inhibit the expression of MYT1 at transcriptional level by scaffolding proteins to establish inhibitory modifications, including DNA methylation, H3K9me3 and H3K27me3 49,50 . Given that the inhibition of linc-321 on MYT1 is transient and histone modifications are supposed to regulate gene expression timely 49 , so we checked the effect of linc-321 on alterations of H3K27me3 and H3K9me3 of MYT1 TSS. We demonstrate that linc-321 can scaffold PRC2 to establish H3K27me3 nearby the TSS region. The role of linc-321 in establishing protein-DNA contacts might reflect a more general mechanism of lncRNA action in identifying genomic binding sites for factors. LncRNAs might act as licensing factors for such events where proteins can assemble in a specific cellular context 30,51 . Further studies are required to understand how linc-321 identifies and binds DNA and the feature of the sequence of linc-321 that makes physical contacts with DNA through canonical lncRNA-DNA interactions such as base pairing or triple helix formation. In the study, we profiled the transcriptome of lincRNAs during the early embryonic development in pig using the scRNA-seq data. The functional lincRNA characterized in pig, linc-321 provides the clues for investigating the strictly regulated process of early embryonic development. Also, considering the conserved principles of early development between human and pig, the study is a basis towards comprehending the role of lincRNA on human early embryos. Declarations Acknowledgements We thank members of the Lab217 for fruitful discussions. Conflict of interest The authors declare no conflict of interests. Funding This work was supported by the National Natural Science Foundation of China, Grant/Award Number: 32272885. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-4250954","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":292710351,"identity":"d87408d6-859a-4c22-9805-fd12e6c262e6","order_by":0,"name":"Xiaogang 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Features of porcine lincRNAs. Comparison of exon number, expression, and transcript length of lincRNAs with protein-coding transcripts were performed. Red: lincRNAs. Blue: protein-coding transcripts.\u003c/p\u003e\n\u003cp\u003ed: Unsupervised hierarchical of the lincRNAs of all the cells from different developmental stages and lineages (28,040 transcripts). Different colors indicate different developmental stages.\u003c/p\u003e\n\u003cp\u003ee: PCA of the lincRNAs of all cells. Different colors indicate different developmental stages and lineages. ICM: inner cell mass; TE: trophoblast.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/f95e9d35a4c51784b89557c2.png"},{"id":55175689,"identity":"d23346d9-7d59-4f3f-9433-f4d3c3a0ab6f","added_by":"auto","created_at":"2024-04-23 16:14:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1596671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elinc-321\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e result in developmental arrest at 1-cell stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Expression patterns of candidate functional lincRNAs based on scRNA-seq data. ICM: inner cell mass; TE: trophoblast.\u003c/p\u003e\n\u003cp\u003eb: Effect of \u003cem\u003elinc-321\u003c/em\u003e and \u003cem\u003elinc-115\u003c/em\u003e knockdown on early embryonic development of pig. 321si: LNA-siRNA targeting on \u003cem\u003elinc-321\u003c/em\u003e; 115si: LNA-siRNA targeting on \u003cem\u003elinc-115\u003c/em\u003e. Neg.: negative control. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003ec: Expression patterns of 42 transcripts from \u003cem\u003elinc-321\u003c/em\u003e locus. Different colors indicate different developmental stages.\u003c/p\u003e\n\u003cp\u003ed: Schematic of \u003cem\u003elinc-321\u003c/em\u003e. Targeting sites of 321si-1 and 321si-2 are indicated. Red arrows: primers for qPCR; green arrows: back part primers for RT-PCR; blue arrows: front part primers for RT-PCR.\u003c/p\u003e\n\u003cp\u003ee: Verification of the effect of \u003cem\u003elinc-321\u003c/em\u003e knockdown on early embryonic development of pig. A scrambled siRNA was used as negative control. Both 321si-1 and 321si-2 injections could significantly reduce the cleavage rate and the blastocyst rate. Different colors indicate different groups indicated. Neg.: negative control. * Indicates significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). The error bars represent s.d.\u003c/p\u003e\n\u003cp\u003ef: Effect of mutant \u003cem\u003elinc-321 \u003c/em\u003eaddback on early embryonic development of pig. The mutant\u003cem\u003e linc-321\u003c/em\u003e with LNA-siRNA targeting site deletion was synthesized (Sango Biotech), and then transcribed\u003cem\u003e in vitro\u003c/em\u003einto RNA. The RNA was injected into porcine MII oocytes, together with 321si-2. Different colors indicate different groups indicated. The developmental rate was calculated by dividing the number of 2-cell embryos by the number of embryos developed to each corresponding stage. Each group performed at least three biological replicates. sd \u003cem\u003elinc-321\u003c/em\u003e: the siRNA targeting site deleted \u003cem\u003elinc-321\u003c/em\u003e; dd \u003cem\u003elinc-321\u003c/em\u003e:\u003cem\u003elinc-321 \u003c/em\u003ewith double deletion of the siRNA targeting site and the sequence predicted to perform the interaction; * Indicates significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The error bars represent s.d.\u003c/p\u003e\n\u003cp\u003eg: Strand-specific RT–PCR (SSRT–PCR) results for \u003cem\u003elinc-321\u003c/em\u003e.\u003cem\u003e Linc-321 \u003c/em\u003eis transcribed from the bottom strand. Primers used in strand-specific reverse transcription are shown in green, while primers used in PCR are shown in red.\u003c/p\u003e\n\u003cp\u003eh: Expression pattern of \u003cem\u003elinc-321\u003c/em\u003e checked by RT-PCR. About 50 1-cell embryos were used for each experiment, and three experimental replicates were performed. FP: front part primers for RT-PCR; BP: back part primers for RT-PCR.\u003c/p\u003e\n\u003cp\u003ei: Expression pattern of \u003cem\u003elinc-321\u003c/em\u003e checked by qPCR. About 50 1-cell embryos were used for each experiment, and three experimental replicates were performed. The error bars represent s.d.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/1291369012b21da61ec327e6.png"},{"id":55175695,"identity":"b0b85208-cd7b-487d-8cbd-ae368db87a88","added_by":"auto","created_at":"2024-04-23 16:14:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1686209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elinc-321\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e results in failure of male and female PEF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: IF staining for β-tubulin of 1-cell embryo at 18h post-IVF. The numbers in the bottom left mean embryos as the figure shown/ total embryos tested.\u003c/p\u003e\n\u003cp\u003eb: Effect of \u003cem\u003elinc-321\u003c/em\u003e knockdown on nuclear morphology at 18h and 24h post-IVF. PNs: pronucleus.\u003c/p\u003e\n\u003cp\u003ec: Nuclear morphology at 18h and 24h post-IVF by \u003cem\u003elinc-321\u003c/em\u003eknockdown.\u003c/p\u003e\n\u003cp\u003ed: Nuclear morphology at 8h post-IVF by \u003cem\u003elinc-321\u003c/em\u003e knockdown. The numbers in the bottom left mean embryos as the figure shown/ total embryos tested.\u003c/p\u003e\n\u003cp\u003ee: EDU staining on 1-cell embryo at 12h post-IVF with or without \u003cem\u003elinc-321 \u003c/em\u003eknockdown. The numbers in the bottom left mean embryos as the figure shown/ total embryos tested.\u003c/p\u003e\n\u003cp\u003ef: IF staining for LAMIN at 8h and 12h post-IVF. The numbers in the bottom left mean embryos as the figure shown/ total embryos tested. Ctrl.: control group; Neg.: embryos injected with scrambled siRNA; \u003cem\u003elinc-321\u003c/em\u003e KD: embryos injected with 321si-2.\u003c/p\u003e\n\u003cp\u003eg: Expression of \u003cem\u003elinc-321\u003c/em\u003e at different time points post-IVF checked by qPCR. About 50 1-cell embryos were used for each experiment, and three experimental replicates were performed. The error bars represent s.d.\u003c/p\u003e\n\u003cp\u003eAll scale bars in the figure, 50 μm.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/19bcf117c6d61b450076386f.png"},{"id":55175701,"identity":"cec37f49-f9e2-4045-9d56-4dfe2b1070d0","added_by":"auto","created_at":"2024-04-23 16:14:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1988915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLinc-321\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e regulates PEF by inhibiting the expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYT1\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Venn diagram of up- and down-regulated genes in 1-cell embryo vs oocyte and 2-cell embryos.\u003c/p\u003e\n\u003cp\u003eb: Quantification of the fluorescence intensity analysis of the protein level of CDC2 after IVF. The protein level of CDC2 was in a low level just after fertilization at 4 h post-IVF and significantly enhanced from 10 h to 12 h post-IVF.\u003c/p\u003e\n\u003cp\u003ec: Immunofluorescence analysis of CDC2 at 18h post-IVF in the control and CDC2 TRIM-Away. The embryos injected with the mixture of TRIM21 mRNA and CDC2 antibody failed to enter mitosis phase.\u003c/p\u003e\n\u003cp\u003ed: Effect of \u003cem\u003elinc-321\u003c/em\u003e knockdown on expression of \u003cem\u003eMYT1\u003c/em\u003etested by qPCR. The embryos were collected at 10h post-IVF. About 50 embryos were used for each experiment, and three experimental replicates were performed. The error bars represent s.d. Different letters indicate very significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003ee and f: Effect of \u003cem\u003elinc-321\u003c/em\u003e knockdown on protein level of MYT1 and p-CDC2 analyzed by WB. The embryos were collected at 10h post-IVF. About 100 embryos were used for each experiment, and three experimental replicates were performed. The error bars represent s.d. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eg: Effect of \u003cem\u003elinc-321\u003c/em\u003e knockdown on expression of MYT1 protein level tested by ELISA. The embryos were collected at 10h post-IVF. About 50 embryos were used for each experiment, and three experimental replicates were performed. The error bars represent s.d. Different letters indicate very significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eh: Knockdown of \u003cem\u003eMYT1\u003c/em\u003e could rescue the deficiency of PEF induced by loss of \u003cem\u003elinc-321\u003c/em\u003e analyzed by IF staining for LAMIN at 12h post-IVF. The numbers in the bottom left mean embryos as the figure shown/ total embryos tested.\u003c/p\u003e\n\u003cp\u003ei: Knockdown of \u003cem\u003eMYT1\u003c/em\u003e could rescue the developmental arrest induced by loss of \u003cem\u003elinc-321\u003c/em\u003e. Each group performed at least three biological replicates. * Indicates significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). \u003cem\u003elinc-321\u003c/em\u003eKD: embryos injected with 321si-2; \u003cem\u003elinc-321\u003c/em\u003eKD \u0026amp; MYT1-Ab: embryos injected with 321si-2 and MYT1 antibody; \u003cem\u003elinc-321\u003c/em\u003e KD \u0026amp; si-\u003cem\u003eMYT1\u003c/em\u003e: embryos injected with 321si-2 and \u003cem\u003eMYT1\u003c/em\u003e siRNA; MYT1-Ab: embryos injected with MYT1 antibody; \u003cem\u003elinc-321\u003c/em\u003e KD at 14h: embryos injected with 321si-2 at 14h post-IVF.\u003c/p\u003e\n\u003cp\u003eAll scale bars in the figure, 100 μm.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/e84ca9ccfae91a74d3b144cf.png"},{"id":55175690,"identity":"b440a28a-9c24-474a-8716-5f49d2b9b6f4","added_by":"auto","created_at":"2024-04-23 16:14:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1012847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLinc-321\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e establishes H3K27me3 to repress expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMYT1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eby recruiting PRC2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Subcellular localization analysis of \u003cem\u003elinc-321\u003c/em\u003e by RNA fractionation and qPCR analysis. The results shown that \u003cem\u003elinc-321\u003c/em\u003e mainly located in the nucleus. \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eXist\u003c/em\u003e act as cytoplasm and nucleus control, respectively. About 200 embryos at 8-10h post-IVF were used for each experiment, and three experimental replicates were performed. The error bars represent s.d.\u003c/p\u003e\n\u003cp\u003eb and c: RNA-FISH for \u003cem\u003elinc-321\u003c/em\u003e in 8-10h post-IVF embryos. The results shown that \u003cem\u003elinc-321\u003c/em\u003e located in the nucleus. The numbers in the bottom left mean embryos as the figure shown/ total embryos tested. The relative quantification of the fluorescence intensity was shown. ** Indicates significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). \u003cem\u003elinc-321\u003c/em\u003e KD was performed as a negative control. Scale bars: 100 μm.\u003c/p\u003e\n\u003cp\u003ed: Effect of \u003cem\u003elinc-321 on \u003c/em\u003eH3K27me3 levels at \u003cem\u003eMYT1\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003epromoter region checked by ChIP-PCR. sd \u003cem\u003elinc-321\u003c/em\u003e:\u003cem\u003e linc-321 \u003c/em\u003ewith LNA-siRNA targeting site deletion; dd \u003cem\u003elinc-321\u003c/em\u003e:\u003cem\u003e linc-321 \u003c/em\u003ewith double deletion of the siRNA targeting site and the sequence predicted to perform the interaction; \u003cem\u003elinc-321\u003c/em\u003e KD: embryos injected with 321si-2. * Indicates significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The error bars represent s.d.\u003c/p\u003e\n\u003cp\u003ee: Interaction of \u003cem\u003elinc-321 \u003c/em\u003ewith PRC2 checked by RIP. RIP was performed using SUZ12 and IgG antibody with about 2,000 1-cell embryos and qPCR was followed to evaluate the enrichment of \u003cem\u003elinc-321\u003c/em\u003e. Three experimental replicates were performed. The error bars represent s.d. Different letters indicate very significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003ef: The possible binding site aligned to\u003cem\u003e linc-321\u003c/em\u003e sequence. The predicted binding sequence in the upstream of \u003cem\u003eMYT1\u003c/em\u003e TSS is marked in black, and the aligned \u003cem\u003elinc-321\u003c/em\u003e sequence is denoted in red.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/0b823eb21b915c1bdcdad4c3.png"},{"id":55175688,"identity":"366370bb-b3af-4575-8bfe-eaa85a278f75","added_by":"auto","created_at":"2024-04-23 16:14:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1037420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of function of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elinc-321\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLinc-321\u003c/em\u003e can facilitate male and female pronuclear envelope fusion by establishing H3K27me3 to repress expression of \u003cem\u003eMYT1\u003c/em\u003e and is indispensable for formation of zygote and early development in pig. The failure of pronuclear envelope fusion and early development induced by loss of \u003cem\u003elinc-321\u003c/em\u003e could be rescued by MYT1 antibody addition.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/7895de8bed632776de5730d4.png"},{"id":55176633,"identity":"782860d5-071c-4c45-9a5b-eda462897378","added_by":"auto","created_at":"2024-04-23 16:22:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3032294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/0971668c-9992-4ba4-ae8d-712414ecc801.pdf"},{"id":55176631,"identity":"26c7aadb-0d9c-4091-8367-34ac53bb669b","added_by":"auto","created_at":"2024-04-23 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16:14:42","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":18814,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable9.docx","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/df7b51222362d06bbb7e8da1.docx"},{"id":55176630,"identity":"de85caf5-7dd6-43f8-9a76-a535a87a4ca3","added_by":"auto","created_at":"2024-04-23 16:22:42","extension":"xlsx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":29498,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable10.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4250954/v1/880310d72401416c3e0736bc.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A novel long intergenic noncoding RNA indispensable for the formation of zygote in pig","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLong noncoding RNAs (lncRNAs), a class of longer than 200 nucleotides (nt) and non-translated endogenous cellular transcripts, have been considered as by-products of transcription without functions. However, they have emerged as new and fundamental transcriptional and post-transcriptional regulators acting at multiple levels of gene expression in the nuclear and/or in the cytoplasmic compartments, and generating an intricate network with RNAs, promoters and enhancers, and chromatin-modifier complexes over the past few years \u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. Considering the versatility of the molecule to operate in different subcellular compartments, via different modes of action and with different target specificity, the annotation and understanding of lncRNAs in various biological process are desirable.\u003c/p\u003e \u003cp\u003eMammalian early embryonic development, from zygote to blastocyst, is a strictly regulated process \u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. Generally, some transcription factors are believed to important for the progression, such as Oct4, which plays an essential role in the development of pluripotent cells in embryo. In addition to the key transcription factors, lncRNAs also participate in a wide variety of the developmental process. For example, promoter-associated noncoding RNAs (pancRNAs), transcribed from bidirectional promoters, have been shown to be indispensable for embryonic development by activation of their partner genes during zygotic genome activation (ZGA) \u003csup\u003e8\u003c/sup\u003e. Moreover, our studies have reported that an endogenous retrovirus (ERV)-associated lncRNA, \u003cem\u003eLincGET\u003c/em\u003e, is identified as one of the earliest known lineage regulators to bias cell fate in 2-cell embryo by promoting the nuclear localization of CARM1 and is essential for embryonic development in mice \u003csup\u003e9,10\u003c/sup\u003e. Our latest research has identified the lncRNA named SAWPA as crucial in the early embryonic development of pigs \u003csup\u003e11\u003c/sup\u003e. The above studies emphasize that lncRNAs are essential regulators of early embryonic development in pigs, which mirrors the complexity and specificity of mammalian development.\u003c/p\u003e \u003cp\u003ePig, as an important livestock, is considered as human disease model and potential organ donor for xenotransplantation in regenerative medicine \u003csup\u003e12\u003c/sup\u003e, and shows conserved principles of early development with human \u003csup\u003e13\u003c/sup\u003e. Therefore, it is urgent to comprehensively understand the molecular mechanism on regulation of embryonic development specific to pig. Mammalian early embryonic development shows broad similarities among mammals, but also reveals crucial species differences in the transcriptional and spatio-temporal regulation. In mice, as a key trophoblast (TE) transcription factor, CDX2 can repress the transcription of OCT4 by binding to the fourth conserved region of the distal OCT4 enhancer to form TE cells \u003csup\u003e14\u003c/sup\u003e. However, we have previously demonstrated that CDX2 can not repress the transcription of OCT4 in porcine early embryos, because of lack of the \u003cem\u003ecis\u003c/em\u003e-acting regulatory region \u003csup\u003e15\u003c/sup\u003e. To further figure out the specificity in pig, we have performed single-cell RNA-seq (scRNA-seq) on porcine early embryos, from 1-cell to early blastocyst (EB), and revealed the molecular features of the first lineage specification, the emergence of pluripotency, the transition of epigenetic landscapes and the dynamics of X-chromosome dosage compensation during the early development \u003csup\u003e16\u003c/sup\u003e. In the study, we profiled the transcriptome of lncRNAs during the early embryonic development in pig using the scRNA-seq data. And we identified a novel nuclear lincRNA, \u003cem\u003elinc-321\u003c/em\u003e, which is essential for the formation of zygote in pig.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Porcine embryo culture and collection\u003c/h2\u003e \u003cp\u003e All experiments were performed according to the guide-lines of The State Key Laboratory Animal Care and Use Committee. The procedure for porcine IVF has been described previously. Briefly, freshly ejaculated sperm-rich fractions were collected from fertile boars. Following short incubation at 39\u0026deg;C, semen was resuspended and washed three times in DPBS supplemented with 0.1% (w/v) BSA via centrifugation at 1500 g for 4 min. Spermatozoa concentrations were measured using a hemocytometer, and the proportion of motile sperm determined. Next, spermatozoa were diluted with modified Tris-buffered medium (mTBM) to an optimal concentration. Cumulus-free oocytes were washed three times in mTBM. Approximately 30 oocytes were inseminated in 50 ml mTBM at a final sperm concentration of 3 \u0026times; 10 5 /ml for 5 h. Embryos were cultured in porcine zygote medium-3 (PZM-3) at 39\u0026deg;C in 5% CO 2 in air. Embryos were collected after IVF at the following time points: 1-cell stage (24 hours), 2-cell stage (40\u0026ndash;45 hours), 4-cell stage (65\u0026ndash;72 hours), 8-cell stage (84\u0026ndash;90 hours), morula stage (108\u0026ndash;115 hours) and blastocyst stage (156\u0026ndash;160 hours). Besides, the oocytes were collected at 42 h \u003cem\u003ein vitro\u003c/em\u003e maturation. For qPCR, about 50 embryos of each stage were used. For α -Amanitin treatment, different concentrations (1\u0026micro;g/ml, 5\u0026micro;g/ml and 10\u0026micro;g/ml) of α -Amanitin (HY-19610, Med Chem Express) was added into culture medium at 6 hours after oocyte activation. To knockdown of \u003cem\u003elinc-321\u003c/em\u003e, we injected about 10 pl 10 \u0026micro;M LNA-siRNA targeting on \u003cem\u003elinc-321\u003c/em\u003e into per MII oocyte, and then performed IVF to obtain embryos. The sequences of siRNAs are shown in Table \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e. For rescue experiment, about 10 pl mixture of 200nM mutant \u003cem\u003elinc-321\u003c/em\u003e from \u003cem\u003ein vitro\u003c/em\u003e transcription was also injected into per MII oocyte. Embryo development was then observed every 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 TRIM-Away assay\u003c/h2\u003e \u003cp\u003epSMPP-mCherry-hTRIM21 vector was purchased from Addgene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.addgene.org/104972/\u003c/span\u003e\u003cspan address=\"https://www.addgene.org/104972/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). mCherry-hTRIM21 was cloned into pCMV6-XL6 expression vector (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.addgene.org/vector-database/5211/\u003c/span\u003e\u003cspan address=\"https://www.addgene.org/vector-database/5211/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using EcoRI and XmaI restriction sites. To make in vitro transcribed mCherry-hTRIM21 mRNA, the template DNA was linearized by AgeIHF digestion. mRNA was generated by in vitro transcription using mMESSAGE mMACHINE T7 Transcription kit (#AM1344; Thermo Fisher Scientific) and purified using RNeasy MinElute Cleanup Kit (#74204; Qiagen). mRNA was aliquoted at a concentration of 2 mg/ml and stored at -80\u0026deg;C. CDC2 antibody (Abcam, ab18) was used. About 10 pl the mCherry-TRIM21 mRNA and CDC2 antibody complex was injected into MII oocyte, and IVF was preformed after injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RNA-seq data quality control, processing and analysis\u003c/h2\u003e \u003cp\u003eSequencing reads obtained from our previous single cell RNA-seq were assessed with the fastX-toolkit (version 0.0.13) to remove short (-l 20) and low quality (-q 20) reads, followed by trimming of the adaptor sequence. The qualified reads were aligned with the Tophat 2 \u003csup\u003e17\u003c/sup\u003e into the porcine reference genome (Suscrofa 10.2.87) by default parameters. The reads on the extracted alignment were constructed with Cufflinks to construct transcripts. The transcripts of all samples were combined and reconstructed into a large transcript file (merged.gtf) using Cuffmerge \u003csup\u003e18\u003c/sup\u003e. Then the expression of genes in each sample were quantified to FPKM by Cuffnorm \u003csup\u003e18\u003c/sup\u003e. For differential expression, we first counted the overlap of reads with genes by htseq-count \u003csup\u003e19\u003c/sup\u003e with the parameter \u0026ldquo;-m union\u0026rdquo;. Next, we compared the two groups using default parameters in R package: DESeq2. A gene was considered significant if the Benjamini and Hochberg\u0026ndash;adjusted Pvalue (Padj) was less than 5% and the fold-change was greater than 2. And we used Short Time-series Expression Miner (v1.3.11) for analysis of gene expression trends.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 LincRNA detection pipeline.\u003c/h2\u003e \u003cp\u003eTo identify lincRNAs in pig, we designed an analysis pipeline to minimize false positives and maximize the number of lincRNA transcripts, including the following five steps: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) we used Cuffcompare to compare our merged transcriptome with annotation in Ensembl databases, and removed potential known transcripts; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) filter transcripts that are shorter than 200 nt; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) select transcripts that are more than 2 exon; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) keep only transcripts that are located at least 500 bp away from any protein-coding genes or house-keeping ncRNAs genes annotated in the Ensembl Sus scrofa10.2 gene set (GTF); (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) filter putative lincRNA transcripts by coding potential using the PLEK \u003csup\u003e20\u003c/sup\u003e, CPC2 \u003csup\u003e21\u003c/sup\u003e and CPAT \u003csup\u003e22\u003c/sup\u003e softwares, which are independent of known annotations and have been proved the best effective lncRNA identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Unsupervised hierarchical clustering\u003c/h2\u003e \u003cp\u003eUsing all expressed genes as input was conducted on all filtered cells by normalized read counts in log 2 scale. The distance method was Euclidean, and the cluster method was ward. D2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence (IF) analysis\u003c/h2\u003e \u003cp\u003eIF analysis of porcine embryos was conducted as previously described \u003csup\u003e8\u003c/sup\u003e. After removal of the zona pellucida with acidic MAN solution, embryos were fixed in 4% PFA for 30 min at room temperature. After three washes for 5 min each in washing solution (0.1% Tween-20, 0.01% Triton X-100 in 1\u0026times; PBS), embryos were permeabilized in normal permeabilizing solution (1% Triton X-100 in 1\u0026times;PBS) for 20 min at room temperature. Embryos were then blocked in blocking solution (1% BSA in 1\u0026times; PBS) for 1 h at room temperature after three washes for 5 min each in washing solution, followed by incubation with primary antibody diluted with blocking solution overnight at 4\u0026deg;C. After three washes for 5 min each in washing solution, embryos were incubated with secondary antibody diluted with washing solution for 1 h at room temperature. After three washes in washing solution, nuclei were stained with Hoechst 33324 for 7 min. Embryos were then mounted on glass slides after three washes. Antibodies in this research: β-Tubulin (Covance, MRB-435p), Lamin (Abcam, ab26300), CDC2 (Abcam, ab18).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Strand-specific RT\u0026ndash;PCR (SSRT-PCR)\u003c/h2\u003e \u003cp\u003eSSRT-PCR was performed using Reverse Transcriptase M-MLV (RNase Hˉ) Kit (TaKaRa). Briefly, mix 1ng\u0026thinsp;~\u0026thinsp;1\u0026micro;g template RNA and 1\u0026micro;L specific primer (F or R) in the tube, and add RNase free H\u003csub\u003e2\u003c/sub\u003eO to 6\u0026micro;L. Holding the template RNA/primer mixture at 70℃ for 10 min, then rapidly cooled on ice for 2 min. Add 2 \u0026micro;L 5\u0026times;M-MLV Buffer, 0.5\u0026micro;L dNTP Mixture (10 mM each), 0.25\u0026micro;L RNase Inhibitor (40 U/\u0026micro;L), 1\u0026micro;L RTase M-MLV (RNase Hˉ) (200 U/\u0026micro;L) and 0.25\u0026micro;L RNase free H\u003csub\u003e2\u003c/sub\u003eO in above tube. After mix the mixture, holding the mixture at 42℃ for 60 min, 70℃ for 15 min. Then perform PCR reaction use above reaction products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 \u003cb\u003eWestern blot (WB)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe protein retrieved from 200 embryos digested with Pierce IP lysis buffer (10 \u0026micro;l/lane) was mixed with 30 \u0026micro;l sample buffer (10 ml; 1.25 ml 0.5 M-pH 6.8-Tris\u0026ndash;HCl, 2.5 ml glycerin, 2 ml 10% SDS, 200 \u0026micro;l 0.5% bromophenol blue, 3.55 ml H2O, and 0.5 ml b-mercaptothion) and incubated for 5 min in boiling water. The samples were separated on SDS\u0026ndash;PAGE with a 5% stacking gel (10 ml; 5.7 ml ddH2O, 2.5 ml 1.5 M pH 6.8 Tris\u0026ndash;HCl, 1.7 ml 30% acrylamide (acryl: bis acryl\u0026thinsp;=\u0026thinsp;29:1), 100 \u0026micro;l 10% SDS, 50 \u0026micro;l 10% ammonium persulfate, and 10 \u0026micro;l TEMED) and a 10% separating gel (10 ml; 4.1 ml ddH2O, 2.5 ml 1.5 M pH 8.8 Tris\u0026ndash;HCl, 3.3 ml 30% acrylamide (acryl: bis acryl\u0026thinsp;=\u0026thinsp;29:1), 100 \u0026micro;l 10% SDS, 50 \u0026micro;l 10% ammonium persulfate, and 5 \u0026micro;l TEMED) at 120 V for 1.5 h and then electrophoretic ally transferred onto a nitrocellulose membrane at 300 mA for 1 h. Membranes were blocked in TBST buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4) containing 3% BSA, for 1 h at RT and then incubated with primary antibody, diluted in TBST containing 1% BSA, overnight at 4\u0026deg;C. After three washes for 10 min each in TBST, the membrane was incubated for 1 h at RT with the secondary antibody diluted in TBST. After three washes for 10 min each, the signals were detected using ECL and films. Antibody used: MYT1(Santa, sc-398299), CDC2-p34(Santa, sc-136014).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Real-time RT-PCR (qPCR) analysis.\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the PureLink\u0026trade; Micro-to-Midi System (Invitrogen) according to the manufacturer\u0026rsquo;s instructions, and reverse transcription was used to generate cDNAs using the PrimeScript\u0026trade; RT Reagent kit (TaKaRa). qPCR was performed using SYBR Premix Ex Taq\u0026trade; (TaKaRa) and the 7500 Real-Time PCR System. The reaction parameters were 95\u0026deg;C for 30 s followed by 40 two-step cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 34 s. Ct values were calculated using Sequence Detection System software, and the amount of target sequence normalized to the reference sequence was alculated as 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e. All primers and probes were designed using Primer Premier 5 (Table \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Subcellular localization analysis\u003c/h2\u003e \u003cp\u003eRNA fractionation Cytoplasmic \u0026amp; Nuclear RNA Purification Kit (Norgen Cat. 21000) was used to prepare the nucleus and cytoplasm separately, according to manufacturers\u0026rsquo; protocols. In brief, 200 embryos were collected to an RNase-free tube and centrifuge at 2,000 RPM for 10 minutes. Add 200ul of ice-cold Lysis Buffer J to the tube. Lyse embryos by vortexing for 15 seconds. Spin lysate for 3 minutes at 16,000 RPM in a benchtop centrifuge. Cytoplasmic RNA is in the supernatant, and nuclear RNA is in the deposition. Collected the supernatant and deposition separately, and add 200ul of Buffer SK and 200ul of 100% ethanol to both RNA fractions to binding RNA to column. Then, wash the column with 400ul Wash Solution A three times. After that, place the column into a fresh Elution tube, and add 50ul of Elution Buffer E to the column. Centrifuge for 2 minutes at 2,000 RPM, followed by 1 minute at 14,000 RPM to collected cytoplasmic RNA and nuclear RNA separately. The following steps are as for Real-time RT-PCR (qPCR) analysis.\u003c/p\u003e \u003cp\u003eRNA-FISH were also performed to confirm the subcellular localization of \u003cem\u003elinc-321\u003c/em\u003e. The probes were labeled by in vitro transcription using DIG RNA Labeling Kit (SP6/T7) (Roche, #11175025910). After removal of the zona pellucida with acidic MAN solution, porcine embryos were incubated in 1\u0026times;PBS containing 6 mg/ml BSA for 15 min. Then, embryos were transferred on coverslips coated with Denhardt\u0026rsquo;s solution (Sigma, #30915-5ML) and dried for 30 min at RT. Embryos were fixed in 3% paraformaldehyde (PFA) for 12 min followed by permeabilization in RNA-FISH permeabilizing solution (0.5% Triton X-100, 10 mM Vanadyl ribonucleotide complex (Sigma, #94742-1ML), in 1\u0026times; PBS) for 6 min on ice. After two washes in 70% EtOH for 5 min each, dehydration was performed in 80%, 95%, twice 100% EtOH, each for 5 min at RT, and the slides were dried for 5 min. The embryos were hybridized in hybridization solution (50% formamide (Sigma), 2\u0026times; SSC, 10% dextran sulfate (Sigma), 10 mM VRC, 2 mg/ml BSA (Sigma)) containing 0.1 nM DIG-labeled RNA probes at 37\u0026deg;C overnight. After three washes for 5 min each in hybridization washing solution (50% formamide, 2\u0026times;SSC) at 42\u0026deg;C and four washes for 5 min each in PBT (1% Tween-20, in 1\u0026times; PBS), we blocked the embryos in blocking solution (10% sheep serum, 0.05% BSA, in 1\u0026times; PBS) for 1 h at RT followed by incubation in antibody hybridization solution (2% sheep serum, 0.05% BSA, anti-DIG antibody (1:200), in 1\u0026times; PBS) for 2\u0026ndash;3 h at RT. After four washes for 5 min each in PBT, embryos were stained with Hoechst 33342 for 7 min. Then, embryos were mounted on glass slides after three washes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Analysis of gene differential expression\u003c/h2\u003e \u003cp\u003eWe compared the two groups using default parameters in R package: DESeq2. A gene was considered significant if the Benjamini and Hochberg\u0026ndash;adjusted Pvalue (Padj) was less than 5% and the fold-change was greater than 2. And we used Short Time-series Expression Miner (v1.3.11) for analysis of gene expression trends.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 ELISA\u003c/h2\u003e \u003cp\u003ePorcine myelin transcription Factor 1 (MYT1) ELISA kit (RJ28474, RENJIEBIO) was used. Capture antibody is pre-coated on the micro titer plate. Firstly, adding standard and samples to wells. Anaylte present was bound by the immobilized antibody. Unbound materials were removed. Secondly, biotin conjugated to detection antibody was added and binded to analyte absorbed on the plate. Unbound ones were washed away. Then, avidin conjugated to HRP was added and binded to biotin absorbed on the plate. Unbound ones were washed away. Finally, add luminal substrate and incubate 10 minutes at 37℃, and read relative light unit value immediately.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 RNA-binding protein immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003eMagnetic Beads Protein G were coated with 5 \u0026micro;g of primary antibody in RIP wash buffer (50 mM Tris\u0026ndash;HCl, pH 7.4, 150 mM sodium chloride, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 0.05% NP-40) containing 5% BSA 3h with rotation at 4℃. Then, we collected 2,000 embryos, and added 100 \u0026micro;L of RIP lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM PMSF/cocktail) and 10 \u0026micro;L of RNase inhibitor (Ambion, AM2694) followed by incubation on ice for 10 min. Next, we centrifuged the RIP lysate at 14,000 rpm for 10 min at 4℃, removed 100 \u0026micro;L of the supernatant and added this to 900 \u0026micro;L of beads-antibody complex in RIP Immunoprecipitation Buffer (860 \u0026micro;L RIP wash buffer, 35 \u0026micro;L 0.5 M EDTA, and 5 \u0026micro;L RNase inhibitor), and incubated this with rotation overnight at 4℃. The residual 10 mL of the supernatant of RIP lysate was treated as input. After washing, treated with proteinase K at 55℃ for 30 min with shaking to digest the protein, followed by RNA extraction from the supernatant; qPCR was then performed to detect \u003cem\u003elinc-321\u003c/em\u003e. Antibody used: SUZ12(Abcam, ab12073); IgG (Millipore, CS200621)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Chromatin Immunoprecipitation (ChIP) assay\u003c/h2\u003e \u003cp\u003eWe collected about 2,000 1-cell embryos and added 100 \u0026micro;l ChIP buffer (150 mM NaCl, 50 mM Tris\u0026ndash;HCl pH 7.4, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM PMSF/cocktail) and 5 \u0026micro;l RNase inhibitor (Ambion, #AM2694) followed by incubation on ice for 10 min. We then harvested 10 \u0026micro;l of lysates used as inputs. The remaining lysates were divided into two parts and incubated with anti-H3K27me3 (Millipore, 07-499) or anti-H3K9me3 (Abcam, ab8898) antibody overnight at 4\u0026deg;C. Next, 10 \u0026micro;l of protein A agarose beads (Novex, #15918014) was added and the mixture was rotated for 4 h at 4\u0026deg;C, followed by microcentrifugation at 900 g for 5 min at 4\u0026deg;C. The beads were washed three times with ChIP buffer for 5 min each. qPCR was then performed. The sequences of primers to detect H3K27me3 level at MYT1 (\u0026plusmn;\u0026thinsp;1.5kb) and GAPDH (\u0026plusmn;\u0026thinsp;1.5kb) promoter region were shown in Table \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 EDU staining\u003c/h2\u003e \u003cp\u003eEDU (10 \u0026micro;M, final concentration) was added to the cultured porcine embryos injected with control-LNA or LNA targeting \u003cem\u003elinc-321\u003c/em\u003e before IVF. After removal of the zona pellucida with acidic MAN solution, embryos were washed two times for 5 min each in washing solution at IVF 12 h. Embryos were then fixed with 3% PFA in 1\u0026times; PBS for 30 min, followed by permeabilization with normal permeabilizing solution. Incorporated EU was detected using the click-iT RNA Alexa Fluor 488 Imaging Kit (Invitrogen, #C10329).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 RNA-Protein Interaction Prediction (RPISeq)\u003c/h2\u003e \u003cp\u003eRNA-Protein Interaction Prediction was performed by online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pridb.gdcb.iastate.edu/RPISeq/index.html\u003c/span\u003e\u003cspan address=\"http://pridb.gdcb.iastate.edu/RPISeq/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS 19.0 for MicroSoft\u0026trade; Windows. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d. The Least Significant Difference method was employed for multiple comparisons. Data were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Transcriptomic profile of lincRNAs during early embryonic development in pig\u003c/h2\u003e \u003cp\u003eSome previous studies have annotated lncRNAs in pig. However, these low-depth datasets are difficult to \u003cem\u003ede novo\u003c/em\u003e annotate lncRNAs accurately due to their low expressions. To comprehensively understand the lncRNA profile during early embryonic development in pig, we annotated lncRNAs using our previous scRNA-seq data. More than 90 single cells from 1-cell embryo to EB were employed to get sequencing libraries. For each sample, we sequenced an average of 20 Gb data \u003csup\u003e16\u003c/sup\u003e, and 15 Gb of clean data were generated, which covered an average of 36\u0026nbsp;million reads. To fully and accurately annotate lncRNAs in porcine early embryos, we combined the data from the same stage samples. We identified 532,564 transcripts in total, and more than 80% known protein-coding genes was confirmed \u003csup\u003e16\u003c/sup\u003e. 41,235 novel lncRNA transcripts from 17,636 loci were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and most of the lncRNAs (68%) were long intergenic noncoding RNAs (lincRNAs; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Consistent with lincRNAs identified in mouse and human, the transcript length and the expression level of the lincRNAs in pig were shorter and lower than those of coding genes, and also the lincRNAs contained less number of exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Unsupervised hierarchical clustering of the lincRNAs (UHC; 28,040 transcripts) grouped the cells according to their developmental stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We found MII oocyte, 1-cell and 2-cell embryos were separated from the embryos at the later stages from 4-cell embryo to EB. In addition, 4-cell embryo was divided from the cluster of 8-cell embryo, morula and EB, which may be related to zygote gene activation (ZGA) occurring at the 4-cell to 8-cell stages in pig. And, the principal-component analysis (PCA) of the lincRNAs exhibited the similar clustering pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The results show that lincRNAs provide a clear visualization of the developmental progress of porcine early embryos.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Knockdown of \u003cem\u003elinc-321\u003c/em\u003e result in developmental arrest at 1-cell stage.\u003c/h2\u003e \u003cp\u003eLots of lincRNAs were found to be highly expressed in 1-cell embryo (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and inhibiting the transcription in 1-cell embryo by α-Amanitin treatment resulted in majority of embryos arresting at the 1-cell stage (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting the transcripts highly expressed in 1-cell embryos, such as the lincRNAs, may be functional during early embryonic development. To identify the functional lincRNAs, we focused on the top 20 highly expressed lincRNAs in 1-cell embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). By injection of LNA-siRNA into oocytes, then used to perform \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF), to knockdown the expression of the lincRNAs, TCONS_00171321 (termed \u003cem\u003elinc-321\u003c/em\u003e) and TCONS_00192115 (termed \u003cem\u003elinc-115\u003c/em\u003e) were found to significantly reduce the ratio of IVF embryos developing to blastocyst \u003cem\u003ein vitro\u003c/em\u003e, and the knockdown of \u003cem\u003elinc-321\u003c/em\u003e also caused the lower ratio to 2-cell stage, indicating the loss of \u003cem\u003elinc-321\u003c/em\u003e caused the developmental arrest at the 1-cell stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). And, by NCBI ORF Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) analysis, only several mini-ORFs were recognized in \u003cem\u003elinc-321\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), confirming its noncoding nature. Thus, in the study, we wanted to show the molecular mechanism of \u003cem\u003elinc-321\u003c/em\u003e on early embryonic development in pig. We first screened all the transcripts from the locus expressing \u003cem\u003elinc-321\u003c/em\u003e. There were 42 transcripts from the locus, and their expression patterns were different during the early development of pig, but only \u003cem\u003elinc-321\u003c/em\u003e was highly expressed in 1-cell embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), suggesting in 1-cell embryo, \u003cem\u003elinc-321\u003c/em\u003e is the most representative and functionally active transcript. The sequences of transcripts from the locus were overlapped (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). To confirm that the developmental arrest at 1-cell stage is contributed by linc321 deletion specifically, we designed a \u003cem\u003elinc-321\u003c/em\u003e-specific LNA-siRNA (321si-2), which targets the second exon of \u003cem\u003elinc-321\u003c/em\u003e and can not target the other transcripts from the locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). We screened the genome-wide profiling of off-targets for 321si-2 by Blastn, and, in addition to the locus expressing \u003cem\u003elinc-321\u003c/em\u003e, another targeting site was found at chr. 3 (Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e), but there was no transcript derived from the locus (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), confirming the negative off-targets of 321si-2. The high efficiency of \u003cem\u003elinc-321\u003c/em\u003e knockdown by the two siRNAs was verified (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), and the consistent result was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee; Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), revealing the functional role of \u003cem\u003elinc-321\u003c/em\u003e on the transition of 1-cell to 2-cell embryos. 2407 bp of the full length of \u003cem\u003elinc-321\u003c/em\u003e with eleven exons, from 89,805,061 to 89,995,64 at chr. 7, was obtained by 5\u0026rsquo; and 3\u0026rsquo; RACE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed; Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). To further avoid the off-target effects by the siRNAs, we deleted the targeting site of 321si-2 in \u003cem\u003elinc-321\u003c/em\u003e and added the siRNA targeting site deleted one (sd \u003cem\u003elinc-321\u003c/em\u003e) back after \u003cem\u003elinc-321\u003c/em\u003e knockdown. As expected, the developmental deficiency was rescued by the addback experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef; Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). To determine the transcription direction of \u003cem\u003elinc-321\u003c/em\u003e, we performed strand-specific RT\u0026ndash;PCR (SSRT\u0026ndash;PCR) analysis and found that \u003cem\u003elinc-321\u003c/em\u003e was transcribed from the bottom strand (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) and qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei) were used to check the expression pattern of \u003cem\u003elinc-321\u003c/em\u003e during the early embryonic development, and the high expression level at the 1-cell stage was verified. We identified a novel lincRNA, \u003cem\u003elinc-321\u003c/em\u003e, and its knockdown resulted in the developmental arrest at 1-cell stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Knockdown of \u003cem\u003elinc-321\u003c/em\u003e results in failure of male and female pronuclear envelope fusion (PEF).\u003c/h2\u003e \u003cp\u003eIt has been demonstrated that the developmental arrest at 1-cell stage may be due to the abnormalities of spindle assembly \u003csup\u003e23\u003c/sup\u003e. To demonstrate the mechanism of the developmental arrest by knockdown of \u003cem\u003elinc-321\u003c/em\u003e, we first checked if spindle assembly was affected by \u003cem\u003elinc-321\u003c/em\u003e knockdown in 1-cell embryo at 18h post-IVF by IF analysis of β-Tubulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Normal spindle was observed in control (21/60) and negative control groups (19/57), but in \u003cem\u003elinc-321\u003c/em\u003e knockdown (\u003cem\u003elinc-321\u003c/em\u003e KD) group, rare formation of normal spindle was observed (2/53). In addition to that, we observed most of 1-cell embryo with two pronuclei (51/53) in \u003cem\u003elinc-321\u003c/em\u003e KD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To further confirm the phenomenon, we tested the nuclear morphology at 18h and 24h post-IVF by DNA staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c) and found the embryos in \u003cem\u003elinc-321\u003c/em\u003e KD group were abnormally arrested at the two pronuclei phase, indicating \u003cem\u003elinc-321\u003c/em\u003e works at an earlier time point. Then, we examined whether \u003cem\u003elinc-321\u003c/em\u003e KD could cause the delay of pronuclear formation. DNA staining on 1-cell embryo at 8h post-IVF was performed, and no significant difference on pronuclear formation among three groups was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), suggesting \u003cem\u003elinc-321\u003c/em\u003e has no effect on pronuclear formation. Furthermore, we checked whether \u003cem\u003elinc-321\u003c/em\u003e functions on DNA replication. By EDU dying on 1-cell embryo at 10h post-IVF, we found that \u003cem\u003elinc-321\u003c/em\u003e KD did not affect DNA replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Given that the pronuclei could sustain to 24h post-IVF in \u003cem\u003elinc-321\u003c/em\u003e KD group, we further detected the role of \u003cem\u003elinc-321\u003c/em\u003e on male and female PEF. IF analysis of LAMIN, the main component of nuclear membrane \u003csup\u003e24\u003c/sup\u003e, was performed. In 1-cell embryo at 8h post-IVF, the nuclear envelope could be observed in both control and \u003cem\u003elinc-321\u003c/em\u003e KD groups. However, at 12h, in control group, PEF had happened, and the zygote was forming, but contrastly, most of \u003cem\u003elinc-321\u003c/em\u003e KD embryos still maintained two pronuclei and could not form zygote (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). To further confirm the function of \u003cem\u003elinc-321\u003c/em\u003e on PEF, we checked its expression levels every 2 hs from 2 h to 16 h post-IVF in 1-cell embryo and found \u003cem\u003elinc-321\u003c/em\u003e was highly expressed specifically at 10 h, just before PEF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). The results show \u003cem\u003elinc-321\u003c/em\u003e is essential for the formation of zygote by facilitating PEF at 1-cell stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eLinc-321\u003c/em\u003e regulates PEF by inhibiting the expression of \u003cem\u003eMYT1\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eLncRNA plays important role on regulation of gene expression by positive and negative ways \u003csup\u003e25,26\u003c/sup\u003e. To demonstrate the mechanism of \u003cem\u003elinc-321\u003c/em\u003e, we tried to screen the genes may be regulated by \u003cem\u003elinc-321\u003c/em\u003e and related to cell cycle. The expression of \u003cem\u003elinc-321\u003c/em\u003e was significantly high in 1-cell embryo, comparing to oocyte and 2-cell embryo. So, we preformed differential expression analysis between 1-cell embryo and oocyte, and 1-cell and 2-cell embryos, to screen the genes with specifically low or high expression level (fold change\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;5) in 1-cell embryo, which were considered as candidates regulated by \u003cem\u003elinc-321\u003c/em\u003e. Among the genes, we found a cell cycle-related gene, MYT1, was significantly low in 1-cell embryo, comparing to oocyte and 2-cell embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Previous studies have shown that MYT1 can phosphorylate CDC2, a major component of Mature Promoting Factor (MPF), to decrease the activity of MPF \u003csup\u003e27,28\u003c/sup\u003e. MPF can promote LAMIN phosphorylation to break down the nuclear envelope, and partial fusion of the male and female pronuclear envelope is the prerequisite for PEF \u003csup\u003e29\u003c/sup\u003e. So, we first check the role of MPF on PEF. We detected the activity dynamics of MPF during the process of PEF by IF detecting the protein level of CDC2. The result showed that MPF activity was in a low level just after fertilization at 4 h post-IVF and significantly enhanced from 10 h to 12 h post-IVF, the time that PEF occur (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). And we observed that the knockdown of CDC2 protein by TRIM-Away resulted in the failure of PEF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The results suggest that MPF may be essential for PEF. So, we checked if \u003cem\u003elinc-321\u003c/em\u003e can regulate the activity of MPF by regulation of \u003cem\u003eMYT1\u003c/em\u003e expression to perform PEF in porcine 1-cell embryo. As expected, the expression of \u003cem\u003eMYT1\u003c/em\u003e was significantly increased by \u003cem\u003elinc-321\u003c/em\u003e KD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Furthermore, the protein level of MYT1 was increased, and phosphorylated CDC2 (p-CDC2), was upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and f). Also, the activity of MYT1 was increased by \u003cem\u003elinc-321\u003c/em\u003e KD detected by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Then, we examined if the knockdown of \u003cem\u003eMYT1\u003c/em\u003e could rescue the abnormalities by \u003cem\u003elinc-321\u003c/em\u003e KD. We observed the injection of MYT1 antibody (MYT1-Ab) could efficiently inhibit the increasing of MYT1 and p-CDC2 by \u003cem\u003elinc-321\u003c/em\u003e KD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and f). PEF occurred normally in \u003cem\u003elinc-321\u003c/em\u003e and MYT1-knockdowned 1-cell embryos at 12h post-IVF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), and the \u003cem\u003ein vitro\u003c/em\u003e development of \u003cem\u003elinc-321\u003c/em\u003e-deficient embryos was rescued by MYT1-Ab and \u003cem\u003eMYT1\u003c/em\u003e siRNA (si-\u003cem\u003eMYT1\u003c/em\u003e) injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh; Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e; Table \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e). We also found the deficiency of MYT1 by MYT1-Ab injection had no effect on the \u003cem\u003ein vitro\u003c/em\u003e development, indicating other nuclear kinases, such as WEE1, may compensate for the loss of MYT1 to inhibit CDC2 by phosphorylating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Furthermore, to confirm that the developmental arrest in \u003cem\u003elinc-321\u003c/em\u003e-deficient embryos was led by the failure of PEF, 321si-2 was injected into 1-cell embryos at 14h post-IVF. As expected, the \u003cem\u003ein vitro\u003c/em\u003e development was not affected by the knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). The result demonstrated \u003cem\u003elinc-321\u003c/em\u003e regulates male and female PEF by inhibiting the expression of \u003cem\u003eMYT1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 \u003cem\u003eLinc-321\u003c/em\u003e establishes H3K27me3 to repress expression of \u003cem\u003eMYT1\u003c/em\u003e by recruiting PRC2 complex.\u003c/h2\u003e \u003cp\u003eTo figure out how \u003cem\u003elinc-321\u003c/em\u003e inhibits the expression of \u003cem\u003eMYT1\u003c/em\u003e, we first investigated the subcellular localization of \u003cem\u003elinc-321\u003c/em\u003e. qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and RNA-FISH (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and c) showed \u003cem\u003elinc-321\u003c/em\u003e was mainly in the nucleus, indicating the inhibition of \u003cem\u003eMYT1\u003c/em\u003e by \u003cem\u003elinc-321\u003c/em\u003e may be at the transcriptional level. It has been demonstrated lncRNA could regulate gene expression by establishment of epigenetic modification \u003csup\u003e3,30\u003c/sup\u003e, and H3K27me3 and H3K9me3 are the main inhibitory modifications. Polycomb Repressive Complex 2 (PRC2) is comprised of EZH2, SUZ12, and EED, and responsible for establishment of H3K27me3 \u003csup\u003e30\u003c/sup\u003e. SETDB1 and SETDB2 are responsible for establishment of H3K9me3 \u003csup\u003e31\u003c/sup\u003e. To test the interaction of the above proteins with \u003cem\u003elinc-321\u003c/em\u003e, RNA-protein interaction was predicted by RPISeq, and we found that the interaction of \u003cem\u003elinc-321\u003c/em\u003e with SUZ12 was positive (PIP-SVM\u0026thinsp;=\u0026thinsp;0.93). Then, RIP was performed using SUZ12 antibody to evaluate the interaction. We found \u003cem\u003elinc-321\u003c/em\u003e could be enriched by SUZ12, comparing to IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Thus, we checked the effect of \u003cem\u003elinc-321\u003c/em\u003e knockdown on the alteration of H3K27me3 at \u003cem\u003eMYT1\u003c/em\u003e promoter region (TSS\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5kb) by ChIP-PCR. The \u003cem\u003eGAPDH\u003c/em\u003e promoter region was also detected as control. The significant loss of H3K27me3 was observed in \u003cem\u003elinc-321\u003c/em\u003e-deficient embryos, and the sd \u003cem\u003elinc-321\u003c/em\u003e could rescued the loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), suggesting \u003cem\u003elinc-321\u003c/em\u003e may establish H3K27me3 to repress the expression of \u003cem\u003eMYT1\u003c/em\u003e. LncRNAs are known to interact with DNA by direct base pairing to assemble protein complex on chromatin at specific targeting sites \u003csup\u003e3,32\u003c/sup\u003e. We found a 20nt sequence of \u003cem\u003elinc-321\u003c/em\u003e was complementary with the sequence at -6,449 to -6,430 of \u003cem\u003eMYT1\u003c/em\u003e TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), indicating the sequence might mediate the lncRNA-DNA interaction. To confirm the interaction, we addback the mutant \u003cem\u003elinc-321\u003c/em\u003e with double deletion of the siRNA targeting site and the sequence predicted to perform the interaction (dd \u003cem\u003elinc-321\u003c/em\u003e) when 321si-2 injection. Unlike sd \u003cem\u003elinc-321\u003c/em\u003e, we found dd \u003cem\u003elinc-321\u003c/em\u003e could not rescue the developmental deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef; Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e), \u003cem\u003eand\u003c/em\u003e ChIP-qPCR show that the enrichment of H3K27me3 signal at MYT1 TSS was also in a significant low level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), indicating the lncRNA-DNA interaction may mediate by the complementary sequences. The results indicate \u003cem\u003elinc-321\u003c/em\u003e could recruit PRC2 and establish H3K27me3 to repress expression of \u003cem\u003eMYT1\u003c/em\u003e at the transcriptional level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn the expanded view of both genomic and transcriptomic analysis, thousands of lncRNAs have been identified in mouse and human \u003csup\u003e33\u0026ndash;35\u003c/sup\u003e. As the majority of loci transcribed into lncRNA display poor conservation \u003csup\u003e33\u003c/sup\u003e, the characterization of lncRNAs in other species is needed. In the study, taking advantage of high-throughput scRNA-seq, we performed comprehensive analysis of lincRNAs during early embryonic development in pig. More importantly, we found \u003cem\u003elinc-321\u003c/em\u003e, a novel nuclear lincRNA highly expressed in 1-cell embryo specifically, could facilitate pronuclear envelope fusion by establishing H3K27me3 to repress expression of \u003cem\u003eMYT1\u003c/em\u003e, and was shown to be indispensable for formation of zygote in pig (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe prediction of lncRNAs in pig is seldom concerned. In our previous study, we have predicted lincRNAs in the early embryos, using the publicly available low input RNA-seq data, and predicted some lincRNAs might play important role on the early developmental process \u003csup\u003e36\u003c/sup\u003e. However, the datasets are collected from different sequencing platforms, and the sequencing depth and biological repeats are inadequate, so the prediction may be not accurate. To more systematically annotate lincRNA in porcine early embryos, in the study, we employed our scRNA-seq data on more than 90 single cells from 1-cell embryo to EB, and the sequencing depth is more than 15G, which will be beneficial to the annotation. To further accurate the annotation, three softwares were combined to predict the coding potential of the transcripts. And, comparing to our previous study \u003csup\u003e36\u003c/sup\u003e, more lincRNAs were identified, which establishes foundation to reveal the functional role of lincRNAs on early embryonic development in pig.\u003c/p\u003e \u003cp\u003eAlthough many lincRNAs have been indicated to play important roles on mammalian preimplantation development \u003csup\u003e26,35,36\u003c/sup\u003e, only a few have been functionally and mechanistically characterized. In the study, we identified a novel nuclear lincRNA, \u003cem\u003elinc-321\u003c/em\u003e, in pig. The full length of \u003cem\u003elinc-321\u003c/em\u003e is 2407bp with eleven exons, from 89,805,061 to 89,995,64 at chr. 7. By careful analysis by qPCR, we found it specifically and transiently expressed at 10h post-IVF. The collection of the 1-cell embryos for scRNA-seq were at 20h post-natural mating. It has been reported that sperm may take 10\u0026ndash;12 hs to enter into the oocyte \u003csup\u003e37,38\u003c/sup\u003e. Thus, the expression patterns of \u003cem\u003elinc-321\u003c/em\u003e checked by scRNA-seq and qPCR are consistent. In general, lncRNAs display low expression levels \u003csup\u003e32,39\u003c/sup\u003e, but the functional ones with defining roles show spatio-temporal expressions in specific tissues and cells, such as \u003cem\u003eLincGET\u003c/em\u003e, which transiently high expressed in the nucleus of one cell of the late 2-cell mouse embryo, to bias the cell fate to ICM. There are 42 transcripts from the locus expressing \u003cem\u003elinc-321\u003c/em\u003e, and their expression patterns are different during the early development of pig. However, given that the development of the deficient embryos is inhibited at the 1-cell stage, we consider \u003cem\u003elinc-321\u003c/em\u003e as the functional transcript, because it is the only transcript highly expressed in 1-cell embryo. Thus, in 1-cell embryo, we believe \u003cem\u003elinc-321\u003c/em\u003e is the most representative and functionally active transcript, and the specific expression pattern of \u003cem\u003elinc-321\u003c/em\u003e indicates its important function on specific events.\u003c/p\u003e \u003cp\u003eEmbryogenesis begins from sperm entering oocyte. The male and female chromosomes are first decondensed and the pronuclei is formed. Then, DNA replication on male and female genomes occurs in the pronuclei, respectively. After that, the male and female pronuclear envelope are partially fused to form the zygote, and the zygote enters mitotic phase. The cleavage of embryo is initiated \u003csup\u003e37,38\u003c/sup\u003e. The formation of zygote happens about at 12h post-IVF in pig \u003csup\u003e38,40\u003c/sup\u003e. \u003cem\u003elinc-321\u003c/em\u003e is just highly expressed before the time point, and we found \u003cem\u003elinc-321\u003c/em\u003e-deficient embryos could not form the zygote, due to abnormal male and female pronuclear envelope fusion. And we oberved linc-321 was mainly in the nucleus. After 12h post-IVF, the nuclear envelop of zygote begins to breakdown, and linc-321 may spread into cytoplasm and be degraded by some nuclease.\u003c/p\u003e \u003cp\u003eIn mammalian embryos, ZGA occurs as early as the 1\u0026ndash;4 cell stage. In mice, the initial ZGA that occurs between S phase of the 1-cell embryo and G1 of the 2-cell embryo is designated as minor ZGA to discriminate it from the burst of transcription that occurs during the mid-to-late 2-cell stage, which is designated as major ZGA \u003csup\u003e41\u003c/sup\u003e. The transcription of minor ZGA is relatively promiscuous, low-level, and genome-wide, and a large number of lncRNAs are produced at that time \u003csup\u003e42,43\u003c/sup\u003e. In the study, the 1-cell embryo of pig analyzed were just around the S phase at 10h post-fertilization, indicating the lincRNAs we identified highly expressed at that time may be derived from minor ZGA, and \u003cem\u003elinc-321\u003c/em\u003e is one of them. In mice, it has been demonstrated that the transient inhibition of minor ZGA results in compromised development beyond the 2-cell stage \u003csup\u003e43\u003c/sup\u003e, and we also found the inhibition of the transcription in 1-cell embryo of pig led to the developmental arrest, suggesting the important role of the lincRNAs derived from minor ZGA on early embryonic development.\u003c/p\u003e \u003cp\u003eGenerally, during oocyte meiosis and early embryonic mitosis, phosphorylation mediated by CDC2, a main component of MPF, is essential for nuclear envelope breakdown, and its phosphorylation resulting in the low activity of MPF can sustain nuclear envelope \u003csup\u003e44,45\u003c/sup\u003e. In oocyte, high activity of MPF is accumulated \u003csup\u003e45\u003c/sup\u003e. After sperm entering into oocyte, the activity of MPF is reduced, and the formation of male and female pronuclei could occur normally, and then the pronuclear envelope are partially fused to form the zygote \u003csup\u003e45\u003c/sup\u003e. However, with the low activity of MPF, the male and female pronuclei could not be breakdown, and PEF could not happen. Thus, the molecular mechanism on PEF with low MPF activity is not clear. In the study, we suggest the increased activity of MPF is critical for the partial breakdown of nuclear envelop of male and female pronuclei, and we demonstrate the transiently high expression of \u003cem\u003elinc-321\u003c/em\u003e to repress the expression of MYT1, a Wee1-related kinase, which can phosphorylate CDC2 on Thr 14 and Tyr 15 to inhibit activity of MPF \u003csup\u003e28\u003c/sup\u003e, can increase the activity of MPF shortly to promote PEF to form the zygote. The loss of \u003cem\u003elinc-321\u003c/em\u003e leads to high expression of \u003cem\u003eMYT1\u003c/em\u003e and the activity of MPF can not be recovered, so the male and female nuclear envelope maintains.\u003c/p\u003e \u003cp\u003eLincRNAs can regulate gene expression by many ways depending on their cellular localization \u003csup\u003e46\u003c/sup\u003e. Cytoplasmic lincRNAs can act as competing endogenous RNAs (ceRNAs) to regulate other RNA transcripts by sponging shared microRNAs \u003csup\u003e4,47\u003c/sup\u003e. And, many nuclear lincRNAs can interact with chromatin-associated proteins, acting as protein scaffolding \u003csup\u003e3,48\u003c/sup\u003e. \u003cem\u003eLinc-321\u003c/em\u003e is mainly in the nucleus, so we suppose it may inhibit the expression of \u003cem\u003eMYT1\u003c/em\u003e at transcriptional level by scaffolding proteins to establish inhibitory modifications, including DNA methylation, H3K9me3 and H3K27me3 \u003csup\u003e49,50\u003c/sup\u003e. Given that the inhibition of \u003cem\u003elinc-321\u003c/em\u003e on \u003cem\u003eMYT1\u003c/em\u003e is transient and histone modifications are supposed to regulate gene expression timely \u003csup\u003e49\u003c/sup\u003e, so we checked the effect of \u003cem\u003elinc-321\u003c/em\u003e on alterations of H3K27me3 and H3K9me3 of \u003cem\u003eMYT1\u003c/em\u003e TSS. We demonstrate that \u003cem\u003elinc-321\u003c/em\u003e can scaffold PRC2 to establish H3K27me3 nearby the TSS region. The role of \u003cem\u003elinc-321\u003c/em\u003e in establishing protein-DNA contacts might reflect a more general mechanism of lncRNA action in identifying genomic binding sites for factors. LncRNAs might act as licensing factors for such events where proteins can assemble in a specific cellular context \u003csup\u003e30,51\u003c/sup\u003e. Further studies are required to understand how \u003cem\u003elinc-321\u003c/em\u003e identifies and binds DNA and the feature of the sequence of \u003cem\u003elinc-321\u003c/em\u003e that makes physical contacts with DNA through canonical lncRNA-DNA interactions such as base pairing or triple helix formation.\u003c/p\u003e \u003cp\u003eIn the study, we profiled the transcriptome of lincRNAs during the early embryonic development in pig using the scRNA-seq data. The functional lincRNA characterized in pig, \u003cem\u003elinc-321\u003c/em\u003e provides the clues for investigating the strictly regulated process of early embryonic development. Also, considering the conserved principles of early development between human and pig, the study is a basis towards comprehending the role of lincRNA on human early embryos.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank members of the Lab217 for fruitful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China, Grant/Award Number: 32272885.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw data of deep sequencing have been deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) as GSE139512.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePefanis, E., et al.: RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. 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Cell. \u003cb\u003e161\u003c/b\u003e, 404\u0026ndash;416 (2015)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"long intergenic noncoding RNA, early embryonic development, formation of zygote, epigenetic regulation, pig","lastPublishedDoi":"10.21203/rs.3.rs-4250954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4250954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLong noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of mammalian early embryonic development. However, in pig, it is seldom investigated. In this study, lncRNAs were predicted using single-cell RNA-seq data on porcine early embryos from oocyte to early blastocyst. We further identified a novel nuclear long intergenic noncoding RNA (lincRNA), \u003cem\u003elinc-321\u003c/em\u003e, which was specifically and highly expressed in 1-cell embryo, and it was indispensable for the formation of zygote and early embryonic development in pig. Its knockdown by locked nucleic acid (LNA)-siRNA could result in developmental arrest at 1-cell stage with failure of pronuclear envelope fusion (PEF). Mechanistically, linc-321 facilitates the recruitment of SUZ12 to the TSS region of MYT1. And this recruitment is instrumental in establishing H3K27me3 modifications, consequently leading to the repression of MYT1 expression. Moreover, the failure of PEF and developmental arrest induced by loss of \u003cem\u003elinc-321\u003c/em\u003e could be rescued by addition of \u003cem\u003elinc-321\u003c/em\u003e with LNA-siRNA targeting site deletion and \u003cem\u003eMYT1\u003c/em\u003e knockdown. Thus, as functional lincRNA characterized in pig, \u003cem\u003elinc-321\u003c/em\u003e provides the clues for investigating the strictly regulated process of early embryonic development.\u003c/p\u003e","manuscriptTitle":"A novel long intergenic noncoding RNA indispensable for the formation of zygote in pig","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-23 16:14:33","doi":"10.21203/rs.3.rs-4250954/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a4a0fa41-dbf4-4ed5-aef9-a7773a169538","owner":[],"postedDate":"April 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":30840302,"name":"Biological sciences/Developmental biology/Embryology"},{"id":30840303,"name":"Biological sciences/Molecular biology/Non-coding RNAs/Long non-coding RNAs"}],"tags":[],"updatedAt":"2024-06-17T07:02:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-23 16:14:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4250954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4250954","identity":"rs-4250954","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}