hnRNPA1 orchestrates multi-layered regulation of SNCA expression

hnRNPA1 orchestrates multi-layered regulation of SNCA expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article hnRNPA1 orchestrates multi-layered regulation of SNCA expression Fedon-Giasin Kattan, Athanasios K Anagnostopoulos, George T Tsangaris, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6294566/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Abnormal accumulation of alpha-synuclein (SNCA) is a hallmark of Parkinson’s disease (PD), yet the molecular mechanisms governing SNCA expression remain incompletely understood. The SNCA 3' untranslated region (3'UTR) plays a critical regulatory role, with linkage disequilibrium suggesting its involvement in sporadic PD. Analysis of SNCA 3'UTR isoforms revealed that longer variants exhibit increased nuclear retention, form nuclear speckles, decay more rapidly, produce less protein, and preferentially localize to the soma rather than axons. Mass spectrometry identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a key regulator of alternative polyadenylation (APA), promoting longer SNCA 3'UTR isoforms. hnRNPA1 selectively binds proximal and distal regions of the SNCA 3'UTR but not the 5'UTR or coding sequence. Functional studies demonstrated that hnRNPA1 enhances SNCA transcription while reducing nucleocytoplasmic shuttling of longer 3'UTR isoforms, destabilizes shorter transcripts, and restricts axonal transport of longer variants. At the protein level, hnRNPA1 decreases SNCA expression by increasing proteasomal degradation and autophagic flux. These findings establish hnRNPA1 as a multifaceted regulator of SNCA, integrating transcriptional and post-transcriptional control via APA, mRNA stability, subcellular transport, and protein turnover. SNCA hnRNPA1 RNA binding proteins alternative polyadenylation 3'UTR mRNA stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION SNCA is an abundant presynaptic protein that acts as a chaperone for SNARE-complex assembly, regulating synaptic neurotransmission. Accumulation of SNCA has been implicated in the development of Parkinson's disease, multiple system atrophy (MSA), and Lewy body dementia (LBD), collectively known as alpha-synucleinopathies. The underlying causes of SNCA-induced neurodegeneration are multifaceted, with misfolded and aggregated forms released from neurons facilitating the spread of pathology in a prion-like manner (reviewed by [ 1 ]). Maintaining physiological levels of SNCA expression is thus essential for neuronal function and viability, yet the molecular mechanisms controlling SNCA expression levels in neurons remain incompletely understood. Post-transcriptional regulation of gene expression is particularly crucial in neurons, given their complex architecture and requirement for local protein synthesis. In eukaryotes, mature mRNA has a tripartite structure consisting of a 5'UTR, a protein-coding region, and a 3'UTR. While the 5'UTR primarily governs translation initiation, the 3'UTR is central to mRNA subcellular localization, stability, translation regulation, and termination. Notably, the mRNAs of presynaptic genes -including SNCA - possess significantly longer 3'UTRs than other transcripts, suggesting an expanded scope for post-transcriptional regulation [ 2 ]. RNA-binding proteins (RBPs) tightly control these processes by binding to specific cis-elements along the RNA sequence. Given the architectural complexity of neurons, characterized by small soma and extensive projections, RBPs play a vital role in the nervous system, orchestrating neurogenesis, neurite outgrowth, synapse formation, and plasticity (reviewed in [ 3 , 4 ]). Among the various RBPs, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is an evolutionarily conserved, highly expressed factor implicated in diverse RNA processing events, including transcription, telomere maintenance, splicing, miRNA maturation, mRNA stability, and translation (reviewed in [ 5 ]). hnRNPA1 possesses two globular RNA recognition motifs for sequence-specific RNA binding and an unstructured low-complexity C-terminal domain, mediating protein-protein interactions involved in stress granule assembly [ 6 ]. This low-complexity domain also exhibits prion-like properties, leading to amyloid aggregation of hnRNPA1, which has been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP), especially when mutations are inherited [ 7 , 8 ]. hnRNPA1 recognizes a variety of RNA sequences with specificity towards a 5'-YAG-3' motif (where Y is C or U) (reviewed in [ 9 ]). APA-mediated lengthening of 3′ UTRs is particularly prevalent in neurons, especially in presynaptic mRNAs like SNCA. This process constitutes a distinct layer of gene regulation [ 10 ] and is considered necessary for the spatial organization of protein synthesis [ 11 ]. Human SNCA mRNA undergoes alternative cleavage and polyadenylation, generating 3'UTR variants of approximately 290, 575, 1075, and 2,529 nucleotides (nt). The 575 and 2,529 nt forms constitute roughly 40% and 35% of SNCA transcripts in the human cortex, respectively [ 12 ]. The longer isoform is upregulated during differentiation [ 13 ] and linked to PD pathology [ 12 ], suggesting its involvement in disease mechanisms. This study investigates how different SNCA 3'UTR isoforms influence protein expression and localization in neurons, focusing on their role in maintaining appropriate SNCA levels. Through an unbiased proteomic approach, hnRNPA1 emerged as a key regulator of SNCA expression via multiple mechanisms. These results provide new insights into the post-transcriptional control of SNCA and highlight potential therapeutic targets for modulating SNCA expression in neurodegenerative diseases. MATERIALS AND METHODS Antibodies For Western blot analysis, the following primary antibodies were used: rabbit polyclonal anti-hnRNPA1 (1:2000, sc-32301, Santa Cruz, CA, USA), mouse monoclonal anti-SNCA (1:1000, sc-7011, Santa Cruz and 1:1000, 610787, BD Transduction), rabbit polyclonal anti-CNOT1 (1:1000, 14276-1-AP, Proteintech Europe, Manchester, UK), rabbit polyclonal anti-CNOT7 (1:1000, 14102-1-AP, Proteintech), rabbit polyclonal anti-PABP1 (1:1000, 10970-1-AP, Proteintech), mouse monoclonal anti-CNOT6 (1:1000, sc-81231, Santa Cruz), mouse monoclonal anti-LAMP2A (1:1000, ab125068, Abcam), mouse anti-GAPDH-HRP conjugated (1:5000, HRP-60004, Proteintech), and anti-puromycin (1:3000, MABE343, Merck Millipore, Darmstadt, Germany). Mouse monoclonal anti-SNCA (1:250, 610787, BD Transduction) and mouse monoclonal anti-FLAG (1:500, F1804, Sigma-Aldrich) were used for immunofluorescence and RNA FISH experiments. Secondary antibodies for Western blot included HRP-conjugated mouse (1:5000, #7076, Cell Signaling Technologies, Danvers, MA, USA) and rabbit (1:5000, #7074, Cell Signaling) antibodies. For immunofluorescence, goat anti-mouse Alexa Fluor 488 (1:500, A11029, Invitrogen) and goat anti-rabbit Alexa Fluor 568 (1:500; A11036, Invitrogen) were used. Immunoprecipitation control antibodies included normal mouse IgG (2 µg, sc-2025, Santa Cruz). Generation of DNA constructs All primers are listed in Supplementary Table S1 . The human hnRNPA1 CDS, SNCA CDS + 3'UTRs, and SNCA 3'UTRs were PCR-amplified using Phusion polymerase (ThermoFisher) from SK-N-SH cDNA. Plasmid construction was performed as follows: hnRNPA1 CDS (KpnI/NotI sites in pENTR-GD), SNCA CDS + 3'UTRs (BamHI/HindIII sites in paavCAG-pre-mGRASP-mCerulean-2A-nls-mCherry) [ 14 ], SNCA 3'UTRs for hybrid constructs (PstI/SmaI sites in EGFP-SNCA-WT) [ 15 ], and SNCA 3'UTRs for luciferase (XhoI/NotI sites in psiCHECK-2, Promega, Madison, USA). Two hnRNPA1 shRNA plasmids targeting all hnRNPA1 transcripts were generated using Block-iT U6 RNAi Entry vector Kit (ThermoFisher). All constructs were sequence-verified by Sanger sequencing (CeMIA SA, Larisa, Greece). Cell culture and transfection SK-N-SH, HEK293A, and Neuro2A cells were maintained in high-glucose DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich). CAD cells were cultured in DMEM/F12 (Sigma-Aldrich) supplemented with 8% FBS. For differentiation experiments, CAD cells were maintained in a medium containing 2% FBS post-transfection. All cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 (ThermoForma, ThermoFisher). Transfections were performed at plating using jetOPTIMUS reagent according to the manufacturer's instructions (Polyplus, Illkirch, France). A 1:1 ratio of DNA (µg) to jetOPTIMUS reagent (µl) was used for plasmid transfections. Cells were transfected with 100 nM siRNA for siRNA experiments using jetPRIME reagent. Transfection efficiency was monitored using EmGFP plasmid, achieving approximately 80% efficiency in SK-N-SH, HEK293A, and Neuro2A cells at 48 h post-transfection. Cells were harvested 24–48 h post-transfection for analysis. Total RNA extraction, cDNA synthesis, and PCR Total RNA was extracted from SK-N-SH cells using TRI Reagent (Molecular Research Centre Inc, Cincinnati, OH, USA). RNA quantity and quality were verified spectrophotometrically (A260/280 > 1.9). For standard cDNA synthesis, RNA was reverse-transcribed with M-MLV reverse transcriptase (ThermoFisher) and random hexamers. For miRNA detection, RNA was polyadenylated using poly(A) polymerase (NEB) before reverse transcription, as previously described [ 16 ]. The resulting cDNA was diluted 11-fold and stored at -80°C. Quantitative PCR was performed using Kapa SYBR Fast Universal 2× qPCR Master Mix (Kapa Biosystems, Roche, Basel, Switzerland) in 96-well PCR microplates (Azenta, Burlington, MA, USA) on a CFX OPUS real-time PCR system (BioRad, Richmond, CA, USA). Negative RT controls were included, and all samples were analyzed in technical triplicates. Data were evaluated using the 2^−ΔΔCT method, normalizing to GAPDH and U6 mRNA levels. Primer sequences are in Supplementary Table S1 . mRNA half-life measurement For mRNA stability, SK-N-SH cells were transfected with hnRNPA1 or control plasmids for 36–48h before treatment with actinomycin D (5 µg/ml; MedChemExpress, Monmouth Junction, NJ, USA). Cells were harvested at 0, 3, 6, and 9h post-treatment, and RNA was analyzed by RT-qPCR. One-phase decay non-linear regression was done in GraphPad Prism (Y0 = 100). Nucleocytosolic fractionation Cell fractionation was performed using ice-cold HLB buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 1 mM EGTA, 0.1% NP40) supplemented with 40U/mL RNAseOUT. After 10 min on ice, lysates were centrifuged at 800×g for 3 min at 4°C to separate cytoplasmic (supernatant) and nuclear (pellet) fractions. Nuclear pellets were washed three times with HLB buffer before resuspension in TRI Reagent. During RNA extraction, nuclear fractions underwent an additional 65°C incubation for 10 min during phase separation to facilitate the release of membrane-bound mRNAs. RPL22-RiboTag assay For each condition, 1×10⁶ SK-N-SH cells were transfected with RPL22-FLAG alone or together with hnRNPA1 or control plasmids. After 48 h, cells were washed twice with ice-cold PBS and harvested in PLB lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl 2 , 0.5% NP-40, 1 mM DTT) supplemented with 1× cOmplete protease inhibitor cocktail and 40U/mL RNAseOUT. After 30-min incubation on ice, lysates were cleared by centrifugation (16,000×g, 10 min, 4°C). Prior to immunoprecipitation, 10% of the supernatant was reserved as input control. FLAG G1 resin (MedChemExpress) was prepared by washing three times with TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). The washed resin was resuspended in TBS containing 40U/mL RNAseOUT and 1× protease inhibitor cocktail. Cleared lysates were combined with prepared resin and incubated for 4 h at 4°C on a rotating mixer. Following incubation, beads were washed three times with NT2 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 0.05% NP-40). RNA was extracted from both beads and input samples using TRI Reagent. In situ hybridization SNCA mRNA was detected using Stellaris® RNA FISH probes (LGC Biosearch Technologies, Hoddesdon, UK). Differentiated CAD cells on poly-D-lysine-coated glass coverslips were washed once with PBS, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed twice with PBS, and permeabilized in 70% ethanol at 4°C for 24h. Following aspiration of ethanol, cells were washed once with Stellaris wash solution A and incubated for 8 h at 37°C with Stellaris hybridization solution containing custom-designed SNCA 3'UTR probes (set of 30 sequences spanning the length of the 3'UTR, conjugated with Quasar™ 570nm fluorophore; 1:100 dilution) and primary anti-synuclein antibody. After hybridization, cells were washed twice with Stellaris wash solution A and incubated with Alexa Fluor™ 488nm secondary antibody (1:500) in wash solution A for 1 h at room temperature. Nuclear staining was performed using DAPI (1 µg/mL) for 3 min, followed by a final wash with Stellaris wash solution B. Coverslips were mounted on microscope slides using SlowFade Gold antifade reagent (Invitrogen) and sealed with nail polish. Images were acquired using a Leica SP5 confocal microscope with a 63× oil immersion objective, maintaining consistent acquisition parameters across all samples. Z-stacks were collected at 0.4 µm intervals, covering the entire cell depth. Affinity pull-down of biotinylated RNA SNCA mRNA domains (5'UTR, CDS, 3'UTR 575 , and 3'UTR 2529 ) were PCR-amplified from SK-N-SH cDNA using domain-specific primers containing the T7 RNA polymerase promoter sequence (5'-AGTAATACACTCACTATAGGG-3') (Supplementary Table S1 ). In vitro transcription was performed in reactions containing 0.75 µg PCR product, 1× T7 buffer, 50 mM DTT, 40U/mL RNAseOUT, NTPs (20 mM A/U/G, 16.3 mM C, 3.7 mM biotin-11-CTP; Roche), and T7 polymerase (Takara Bio) at 42°C for 2 h. Template DNA was removed using RNase-free DNAase I (NEB) for 20 min at 37°C, followed by LiCl precipitation of RNA. RNA probes were prepared in structure buffer (10 mM Tris pH 7.0, 0.1 M KCl, 10 mM MgCl 2 ), heated to 90°C for 2 min, cooled on ice for 2 min, and equilibrated at room temperature for 20–30 min for proper folding. One postnatal day 3 mouse brain or 10 7 SK-N-SH cells were used for each pull-down. Cell lysates were prepared in ice-cold NT2 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 0.05% NP-40) supplemented with protease inhibitors. Following a 45-min rotation at 4°C, lysates were centrifuged at 16,000×g for 10 min at 4°C. RNA-protein binding was performed by incubating lysates with 2 µg of RNA probes for 2 h at room temperature, followed by adding pre-washed streptavidin magnetic beads for another 2 h. After three NT2 buffer washes, bound proteins were eluted using NT2 buffer containing 1× Laemmli buffer (250 mM Tris-HCl pH 6.8, 6% SDS, 30% β-mercaptoethanol, 40% glycerol, 0.005% bromophenol blue) at room temperature for 15 min followed by 10-min boiling. Peptide Generation and 1-D nanoLC-MS/MS analysis Protein extraction and peptide generation were performed according to established protocols [ 17 ]. Briefly, proteins were extracted in 7 M urea buffer containing 80 mM triethyl ammonium bicarbonate (TEAB), followed by 30-min sonication in a water bath. Reduction and alkylation were performed using 10 mM dithiothreitol and 55 mM iodoacetamide, respectively. LC-MS/MS analysis was performed using an LTQ Orbitrap Elite mass spectrometer interfaced with a Dionex Ultimate 3000 HPLC system (Thermo Scientific, Rockford, IL, USA) [ 17 ]. Peptide separation was achieved using two Thermo Scientific columns in series: a PepMap RSLC C18 column (100 Å, 3-µm-bead-packed 15-cm column) followed by a PepMap RSLC C18 column (100 Å, 2-µm-bead-packed 50-cm column). Peptides were eluted at a flow rate of 3 nL min-1 using mobile phase A (0.1% formic acid in water) and mobile phase B (99% acetonitrile, 0.1% formic acid). The gradient program was optimized: 2.0% B for 10 min, linear increase from 2.0–35.0% B over 325 min, increase to 80.0% B and hold for 10 min, return to 2.0% B, and re-equilibrate for 10 min. Mass spectrometric data were acquired using a data-dependent acquisition strategy. Full MS scans were acquired in the Orbitrap at a resolution of 60,000 (at m/z 400) with a scan range of 250–1250 m/z and a maximum injection time of 250 msec. The top 20 most intense precursor ions from each MS scan were selected for fragmentation using higher energy collision dissociation (HCD) with a normalized collision energy of 36%. MS/MS spectra were acquired in the Orbitrap at a resolution of 15,000 with a maximum injection time of 120 msec. Dynamic exclusion parameters included one repeat count, 30-sec repeat duration, 120-sec exclusion duration, and a ± 0.6 m/z low and ± 1.6 m/z high exclusion mass width. Lock mass calibration was performed using m/z 445.120025 as an internal standard. Raw data files were processed using the SEQUEST algorithm within Proteome Discoverer™. MS/MS spectra were searched against the appropriate protein database using the following parameters: parent ion mass tolerance of 20 ppm, fragment mass tolerance of 0.05 Da, trypsin as the cleavage enzyme allowing up to 2 missed cleavages, cysteine methylthio as a fixed modification, and methionine oxidation as a variable modification. Peptide identifications were filtered to 1% false discovery rate (q-value ≤ 0.01) using the percolator algorithm with Delta Cn maximum set at 0.05. The minimum peptide length was set to six amino acids. Immunoprecipitation of RNP complexes (RIP) For RNA-protein complex identification, protein A/G Sepharose beads (sc-2003, Santa Cruz Biotechnology) were pre-coated with 2 µg of anti-hnRNPA1 or anti-IgG antibody overnight at 4°C in NT2 buffer containing 5% BSA under constant agitation. Beads were washed three times with NT2 buffer. Cell extracts were prepared from 10 7 SK-N-SH cells using ice-cold PLB lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl 2 , 0.5% NP-40, 1 mM DTT) supplemented with 1× cOmplete protease inhibitor cocktail and 40U/mL RNAseOUT. Following 30-min incubation at 4°C, debris was removed by centrifugation at 16,000×g for 10 min. Cell extracts were combined with antibody-coated beads and incubated at 4°C for 4 h on a rotating mixer, with a small aliquot reserved as input control. After three NT2 buffer washes, proteins were digested using proteinase K, followed by RNA extraction using Tri Reagent. Preparation of whole protein extracts and Western blotting Whole-cell lysates were prepared using ice-cold RIPA buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM EDTA, 1% Triton X-100, 0.16% Sodium Deoxycholate, 0.16% SDS) supplemented with 1× cOmplete protease inhibitor cocktail. Following 30-min incubation on ice, lysates were centrifuged at 16,000×g for 30 min at 4°C. Supernatants were stored at -80°C until use. Protein concentration was determined using the Bradford assay according to the manufacturer's protocol (BioRad). For immunoblotting, equal amounts of protein extracts were combined with 6× SDS sample buffer (375 mM Tris pH 6.8, 10% SDS, 50% glycerol, 10% β-mercaptoethanol, 0.03% bromophenol blue), heated at 100°C for 5 min, and separated by 12% or 15% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose membranes (Amersham/Merck) and blocked with 5% non-fat milk in TBS-T (TBS containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were diluted in TBS-T and incubated overnight at 4°C, followed by HRP-conjugated secondary antibodies (1 h, room temperature). Immunoreactive bands were visualized using Clarity or Clarity Max ECL reagents (BioRad) and imaged using a Fusion FX6 system (Vilber, Marne-la-Vallée, France). Densitometric analysis was performed using Fiji software (NIH), with ACTD or GAPDH as the normalization controls. Surface sensing of translation (SUnSET) method Protein synthesis rates were analyzed using the SUnSET method, as described by Schmidt et al. [ 18 ]. At 48 h post-transfection, SK-N-SH cells were treated with 1 µM puromycin (P8833, Sigma-Aldrich) for 30 min. Cells were harvested in ice-cold RIPA buffer, and protein extracts were processed for Western blotting as described above. Puromycin-labeled peptides were detected using an anti-puromycin antibody (1:3000, MABE343, Merck Millipore), followed by HRP-conjugated secondary antibody and ECL detection. Luciferase reporter assay Luciferase activities were measured 48 h post-transfection using the Dual-Luciferase Assay System (Promega) according to the manufacturer's protocol. Measurements were performed in a Lumat LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). Renilla luciferase (target) expression was normalized to Firefly luciferase (internal reference control), with results presented as a percent ratio of Renilla to Firefly activity. Gene Ontology Analysis Gene Ontology (GO) analyses for 'Molecular function' and 'Biological process' were conducted using the database for annotation, visualization, and integrated discovery (DAVID) with default parameters [ 19 ]. Statistical analysis All experiments were performed with at least three biological replicates, and data are presented as mean ± standard deviation (SD). Statistical significance between two groups was determined using two-tailed Student's t-test. Multiple comparisons were analyzed using one-way ANOVA followed by Dunnett's post hoc test. Statistical significance was set at p < 0.05. All statistical analyses were performed using GraphPad Prism version 8.0.0 (San Diego, California, USA). RESULTS Differential nuclear retention and distribution of SNCA 3'UTR variants The relative abundance of the longest SNCA transcript variant was approximated in SK-N-SH neuroblastoma and HEK293A cells, which express high levels of endogenous SNCA. RT-qPCR analysis using primers spanning the first segment of SNCA 3'UTR (detecting all isoforms) and the distal region of the longest SNCA transcript (3'UTR 2529 ) revealed that this extended variant comprises roughly 30% of total SNCA mRNAs as reported in the human cortex, based on a 2 Ct value difference between isoforms. Analysis of nuclear and cytosolic fractions from SK-N-SH cells demonstrated distinct compartmentalization patterns among SNCA variants. The fractionation protocol efficacy was validated using intronic SNCA primers (Fig. 1 a). Quantification using SNCA 3'UTR proximal and distal primers revealed a four-fold higher nuclear abundance of the long transcript compared to total transcripts (p < 0.05), indicating extended nuclear retention (Fig. 1 a). These findings were corroborated using CNS catecholaminergic CAD cells, which were selected for their lack of endogenous SNCA expression. Following transfection with SNCA constructs containing the four main 3'UTR length variants, RNA FISH analysis demonstrated enhanced nuclear localization of 3'UTR 2529 transcripts, with longer variants (3'UTR 1075 or 3'UTR 2529 ) forming twice as many nuclear speckles compared to shorter variants (3'UTR 290 or 3'UTR 575 ) (p < 0.05, Fig. 1 b, c). These findings establish distinct nuclear retention patterns among SNCA variants, with longer transcripts showing enhanced nuclear accumulation and speckle formation. Longer SNCA 3'UTR variants show reduced stability and translation efficiency Transcript stability analysis in actinomycin D-treated SK-N-SH cells revealed that the long transcript exhibited approximately 25% faster decay (p < 0.05) compared to total transcripts across all time points (Fig. 1 d). Similar stability patterns were observed in CAD cells expressing SNCA constructs with 3'UTR 290 or 3'UTR 2529 (data not shown). The impact of 3'UTR length on translation was evaluated through three complementary approaches. First, CAGG-driven SNCA constructs were expressed in Neuro2a cells, chosen for their absence of endogenous SNCA expression. Western blot analysis demonstrated progressively reduced protein production with increasing 3'UTR length (Fig. 1 e). Second, fluorescence microscopy of CAD cells co-expressing EGFP CDS-SNCA 3'UTR hybrids containing the four main SNCA 3'UTR length variants with RFP as normalization control showed diminished EGFP fluorescence proportional to 3'UTR length (Fig. 1 f). Third, dual-luciferase assays in SK-N-SH cells revealed that 3'UTR 2529 reduced luciferase activity by 30% compared to 3'UTR 575 (Fig. 1 g). Consistent with reduced translation efficiency, RiboTag analysis in SK-N-SH cells expressing flagged RPL22 showed significantly decreased ribosomal association of long SNCA transcripts compared to total SNCA mRNA (p < 0.001) (Fig. 1 h). Together, these approaches demonstrate that more extended SNCA 3'UTR variants exhibit reduced stability and translational efficiency. Differential subcellular localization of SNCA variants Combined RNA FISH and immunofluorescence analysis in differentiated CAD cells revealed distinct subcellular distribution patterns among SNCA variants. Longer transcripts showed increased cytoplasmic speckle formation, with 3'UTR 2529 displaying 70% more speckles than 3'UTR 290 (p < 0.05) (Fig. 1 i). Moreover, longer variants (3'UTR 1075 or 3'UTR 2529 ) exhibited preferential somatic localization, with 30% reduced axonal protein levels (p < 0.01) compared to shorter variants (Fig. 1 j). These findings demonstrate that SNCA 3'UTR length influences the spatial distribution of both transcripts and proteins, with longer variants showing distinct subcellular localization patterns that could impact local SNCA protein availability. hnRNPA1 preferentially associates with SNCA 3'UTR and modulates alternative polyadenylation To identify RBPs that interact with SNCA 3'UTR and regulate APA, biotinylated RNA spanning the 2,529 nt SNCA 3'UTR was synthesized and incubated with total brain lysates from postnatal day three mouse brains (Fig. 2 a). RNP complexes were isolated using streptavidin-coated magnetic beads and analyzed by nano LC-MS/MS as an initial screening approach (Supplementary Table S2 ). Differential analysis of proteins specifically enriched on SNCA 3'UTR beads compared to control beads was performed using GOTERM analysis in DAVID [ 19 ]. This revealed enrichment for 'nucleic acid binding' (27 proteins, 20.5%, Benjamini 4.2 × 10 − 12 ), 'RNA binding' (29 proteins, 22.0%, Benjamini 5.6 × 10 − 9 ), and 'double-stranded DNA binding' (12 proteins, 9.1%, Benjamini 3.4 × 10 − 6 ) activities (Fig. 2 b). The most enriched biological processes included 'mRNA processing' (17 proteins, 12.9%, Benjamini 3.2 × 10 − 6 ), 'RNA splicing' (14 proteins, 10.6%, Benjamini 2.8 × 10 − 5 ), and 'mRNA stabilization' (5 proteins, 3.8%, Benjamini 3.3 × 10 − 2 ) (Fig. 2 c). All RBPs detected in the SNCA 3'UTR 2529 pull-down analysis are presented in Fig. 2 d, as even proteins showing some background binding could have physiologically relevant RNA interactions. To investigate their role in SNCA alternative polyadenylation regulation, 17 classical RBPs containing canonical RNA recognition motifs were selected from the identified RNA-binding proteins, cloned, and individually expressed in HEK293A cells. RT-qPCR analysis after 48 h revealed that hnRNPA1 most potently induced 3'UTR lengthening, doubling the ratio of long to total SNCA transcripts (p < 0.01) (Fig. 2 e). Analysis using ENCORI's platform showed strong anticorrelation between hnRNPA1 and SNCA mRNA levels in lower-grade brain gliomas, suggesting a regulatory relationship (Fig. 2 f). Given hnRNPA1's high expression levels, crucial role in RNA processing, and association with neurodegenerative diseases including familial ALS through its prion-like properties, it was selected for comprehensive analysis of its impact on SNCA expression. The interaction between hnRNPA1 and endogenous SNCA mRNA was validated through RNA immunoprecipitation in SK-N-SH cells under native conditions (without UV crosslinking). Real-time RT-PCR analysis of RNA isolated from hnRNPA1 and control IgG immunoprecipitates showed six-fold enrichment of SNCA mRNA in hnRNPA1 complexes, using ACTB mRNA, which contaminated all samples at low levels, as a normalization control (Fig. 2 g). To map hnRNPA1 binding sites along SNCA mRNA, four biotinylated RNA transcripts were generated spanning the 5'UTR, CDS, proximal (575 nt), and distal (2,000 nt) portions of the 3'UTR (Fig. 2 h, schematic). RNA-protein complexes were isolated from SK-N-SH lysates using these transcripts. Western blot analysis revealed that hnRNPA1 exclusively bound both proximal and distal portions of the SNCA 3'UTR, consistent with multiple predicted binding sites throughout these regions (Fig. 2 h). Collectively, these findings establish hnRNPA1 as a key regulator of SNCA mRNA processing through direct binding to multiple sites within the 3'UTR, with a particular impact on alternative polyadenylation and transcript variant distribution. hnRNPA1 enhances SNCA transcription Given hnRNPA1's established role in transcriptional regulation [ 20 , 21 ], its impact on SNCA transcription was evaluated through gain- and loss-of-function experiments in SK-N-SH cells. Following 48 h of hnRNPA1 overexpression, RT-qPCR analysis of nuclear RNA revealed increased SNCA pre-mRNA and mRNA levels across multiple regions: intron 3-exon 4 (61%, p < 0.05), intron 5-exon 6 (35%, p < 0.05), and proximal exon 6 (3'UTR) (11%, p < 0.05) (Fig. 3 a-c). Conversely, simultaneous expression of two hnRNPA1 shRNA plasmids for 48 h resulted in only marginal decreases in SNCA (pre-)mRNA levels, suggesting that reduced hnRNPA1 expression has a limited impact on SNCA transcription. These results collectively establish hnRNPA1 as a positive regulator of SNCA transcription, with elevated hnRNPA1 levels sufficient to enhance SNCA mRNA expression. hnRNPA1 modulates SNCA transcript distribution and local translation The role of hnRNPA1 in SNCA transcript localization was investigated, given its known nucleocytoplasmic shuttling capabilities. In SK-N-SH cells, modulating hnRNPA1 levels for 48 h did not affect the nuclear export of total SNCA mRNA (Fig. 4 a). However, hnRNPA1 overexpression specifically reduced the nuclear export of long SNCA transcripts by 47% (p < 0.05), while its depletion enhanced nuclear export by 38% (p < 0.05) (Fig. 4 b). The impact of hnRNPA1 on SNCA ribonucleoprotein (RNP) complex formation and transport was further examined in differentiated CAD cells chosen for their extensive neuritic outgrowth. Co-expression of hnRNPA1 with SNCA constructs containing different 3'UTR lengths, followed by in situ hybridization after 24 h, revealed distinct effects on RNP distribution. hnRNPA1 increased nuclear speckles for the shortest SNCA RNP complexes (370nt: 23%, p < 0.001) (Fig. 4 c) and enhanced cytoplasmic speckle formation for shorter SNCA RNPs (370nt: 143%, p < 0.05; 570nt: 232%, p < 0.05) (Fig. 4 d). Additionally, hnRNPA1 significantly reduced axonal levels of longer SNCA transcripts (570nt: -60%, p < 0.001; 1075nt: -56%, p < 0.01; 2550nt: -62%, p < 0.05) (Fig. 4 e), with corresponding decreases in axonal protein levels (570nt: -38%, p < 0.01; 2550nt: -31%, p < 0.05) (Fig. 4 f). These findings demonstrate that hnRNPA1 differentially regulates SNCA transcript distribution based on 3'UTR length, particularly affecting longer transcripts through reduced nuclear export, altered RNP complex formation, and decreased axonal localization and translation. hnRNPA1 regulates SNCA mRNA stability and alternative polyadenylation The role of hnRNPA1 in steady-state SNCA mRNA levels was examined. Plasmid-mediated overexpression of hnRNPA1 in SK-N-SH and HEK293A cells for 48 h reduced total levels of endogenous SNCA mRNA by approximately 30% (p < 0.05) in both cell lines. Conversely, the expression of the long transcript increased by approximately 40% (p < 0.05) in both cell lines. The ratio of the long over total SNCA transcripts was approximately 1.6 for both cell lines (p < 0.005 for SK-N-SH and p < 0.01 for HEK293), indicating that excess hnRNPA1 induces 3'UTR lengthening and destabilizes SNCA transcripts (Fig. 5 b-d). Notably, silencing hnRNPA1 expression by overexpressing hnRNPA1 shRNA plasmids for 48 h also decreased total SNCA transcript levels by 48% (p < 0.01) in SK-N-SH and 37% (p < 0.05) in HEK293 cells. Lower hnRNPA1 expression significantly reduced the long SNCA transcript levels in HEK293 (35%, p 0.05) cells. Following hnRNPA1 downregulation, the long-over-total SNCA transcripts ratio did not change in either cell line (Fig. 5 f-h). To understand the mechanism behind these changes in steady-state levels, the stability of SNCA transcripts was analyzed. Forty-eight hours after transfecting SK-N-SH cells with plasmids overexpressing or silencing hnRNPA1, actinomycin D treatment revealed that hnRNPA1 overexpression significantly decreased the half-life of total SNCA mRNA (Fig. 6 a). In contrast, silencing hnRNPA1 increased total SNCA mRNA expression at most time points (Fig. 6 b). Importantly, hnRNPA1 levels did not significantly affect the stability of the SNCA transcripts with the long 3'UTR (Fig. 6 c, d). These experiments demonstrate that hnRNPA1 destabilizes SNCA transcripts with shorter 3'UTRs, significantly downregulating total SNCA mRNA levels. The regulatory effect is diminished on the longer SNCA transcripts, possibly due to their increased nuclear localization following hnRNPA1 overexpression and interference from additional RBPs bound to the distal segment of SNCA's 3'UTR. The mechanism by which hnRNPA1 induces SNCA mRNA degradation was then investigated. Since previous studies have shown that hnRNPA1 can enhance the transcription or maturation of specific miRNAs [ 22 , 23 ], and miR-7 and miR-153 are known to regulate SNCA mRNA expression [ 24 ], the possibility that hnRNPA1 either enhances these miRNAs or promotes their recruitment on SNCA 3'UTR was examined. RT-qPCR analysis of miR-7 and miR-153 expression was performed on RNA extracted from SK-N-SH cells transfected with the plasmids overexpressing or silencing hnRNPA1. Mature miR-7 levels were increased by approximately 37% (p < 0.05), while miR-153 expression was unaffected by hnRNPA1 levels (Fig. 6 e, f). Next, a plasmid co-expressing miR-7 and miR-153 was co-transfected with the plasmids overexpressing or silencing hnRNPA1 in SK-N-SH cells. Forty-eight hours later, RT-qPCR analysis of SNCA mRNA expression revealed that hnRNPA1 overexpression or silencing did not alter total or long SNCA transcripts degradation mediated by miR-7/153 (Fig. 6 g, h), suggesting that hnRNPA1 does not regulate the recruitment of miRISC-7/153 complexes on SNCA mRNA and that it is dispensable for their function. The CCR4–NOT (carbon catabolite repression 4-negative on TATA-less) complex, the major deadenylase in mammals, is formed by CNOT1 that acts as a scaffold to about seven subunits of which CNOT6 and CNOT7/CAF1 are the catalytic members. To investigate if hnRNPA1 directly recruits this complex on SNCA 3′UTR, the biotinylated SNCA 3'UTR 2529 was incubated with total lysates from SK-N-SH cells expressing varying quantities of hnRNPA1. Western blot analysis showed that hnRNPA1 did not pull CNOT1 or CNOT7 on SNCA 3′UTR (Supplementary Fig. 1). As the assay does not favor third-order interactions, the possibility that hnRNPA1, via interaction with another RBP, recruits CCR4–NOT to SNCA mRNA cannot be excluded [ 25 , 26 ]. PABPC1 binds to mRNAs' poly(A) tails, protecting them from shortening. To test if hnRNPA1 affected SNCA levels by altering PABPC1 levels, hnRNPA1 expression was modulated in SK-N-SH cells. Immunoblotting revealed that hnRNPA1 overexpression significantly decreased PABPC1 levels (31%, p < 0.01), while lowering hnRNPA1 levels did not have an effect (Fig. 6 i). These data demonstrate that hnRNPA1 regulates SNCA mRNA levels through multiple coordinated mechanisms. While promoting the decay of shorter transcripts in the cytoplasm through increased miR-7 expression and reduced PABPC1-mediated protection, the enhanced nuclear retention of longer transcripts shields them from these cytoplasmic degradation mechanisms. The observation that silencing hnRNPA1 leads to decreased steady-state SNCA mRNA levels despite increased transcript stability suggests adaptation to prolonged hnRNPA1 depletion, where cells establish a new equilibrium with fewer but more stable transcripts. Subsequently, the mechanism by which hnRNPA1 drives SNCA mRNA alternative polyadenylation was investigated. In eukaryotes, cleavage and polyadenylation require specific interactions between cis-acting sequences and their corresponding protein complexes (Fig. 7 a). The core polyadenylation signal (PAS), typically AAUAAA or AUUAAA, is recognized by CPSF30/CPSF4 and WDR33 of the polyadenylation specificity factor (CPSF) complex. Downstream of the PAS, CSTF64/CSTF2 of the cleavage-stimulating factor (CSTF) complex binds to a GU-rich downstream element (DSE). The interaction between CPSF and CSTF complexes is crucial for determining cleavage site selection. Additionally, the cleavage factor I (CFIm) complex, comprising NUDT21/CPSF5 and CFIm68/CPSF6, binds to U(G/A)UA motifs upstream of the PAS to facilitate cleavage site recognition. A fourth regulatory element, rich in G nucleotides and located downstream of the DSE, may also participate in polyadenylation through yet unidentified factors. To investigate whether hnRNPA1 influences APA by interfering with these core processing complexes, biotinylated SNCA 3'UTR 2529 was incubated with total lysates from SK-N-SH cells expressing increasing amounts of hnRNPA1. While the association of hnRNPA1 with SNCA 3'UTR increased as expected, the binding of CPSF, CSTF, and CFIm complexes remained unchanged (Fig. 7 b), suggesting that hnRNPA1 does not directly compete with core APA machinery. Another potential mechanism involves the spliceosomal component U1 snRNP. Previous studies have shown that hnRNPA1 can directly interact with U1 snRNP [ 27 ], and reducing U1 snRNA levels can activate proximal polyadenylation sites [ 28 ]. Furthermore, U1 snRNP complexed with RNA polymerase II can influence co-transcriptional recruitment of 3′ processing factors, similar to mechanisms proposed for other RNA-binding proteins [ 29 , 30 ]. RT-qPCR analysis following U1 snRNA depletion with two different siRNAs showed that despite 80% reduction in U1 snRNA levels, the ratio of long-to-total SNCA transcripts showed a non-significant 10% increase, indicating that U1 snRNP is not essential for regulating SNCA APA site selection (data not shown). The role of transcription elongation rate in APA site selection was also examined, as increased pausing of RNA polymerase II (Pol II) downstream of an early PAS can enhance cleavage and polyadenylation at that specific site [ 11 ]. This mechanism is particularly relevant since proximal sites are encountered first by the APA machinery. hnRNPA1 is known to enhance transcription elongation by sequestering 7SK RNA, a repressor of the transcription elongation factor P-TEFb [ 31 , 32 ], and its depletion leads to promoter-proximal pausing of RNA polymerase II on P-TEFb-dependent genes [ 33 ]. Treatment of hnRNPA1-expressing SK-N-SH cells with DRB for 2 h, which blocks P-TEFb kinase binding to Pol II, followed by DRB removal, revealed that control cells showed the expected progressive increase in SNCA transcripts. In contrast, hnRNPA1-transfected cells showed minimal response to DRB (Fig. 7 c). This resistance to DRB treatment suggests maintained P-TEFb engagement with Pol II in the presence of hnRNPA1, supporting a model where hnRNPA1 promotes transcription elongation and consequently favors distal polyadenylation site usage in SNCA mRNA processing. These findings demonstrate that hnRNPA1 promotes distal polyadenylation site usage primarily through enhanced transcription elongation rather than direct interference with the core APA machinery or U1 snRNP-dependent mechanisms. hnRNPA1 regulates SNCA protein expression through multiple pathways The impact of hnRNPA1 on SNCA protein expression was evaluated in SK-N-SH and HEK293A cells. Western blot analysis revealed that hnRNPA1 overexpression decreased total SNCA protein levels by 34% (p < 0.005) in SK-N-SH and 57% (p < 0.001) in HEK293A cells (Fig. 8 a). To determine whether this decrease resulted from reduced mRNA levels or translation inhibition, ribosomal association of SNCA mRNA was assessed using the RPL22-RiboTag assay in SK-N-SH cells. RNA-IP analysis showed that hnRNPA1 overexpression did not alter the association of total SNCA transcripts with ribosomes (Fig. 8 b), suggesting that excess hnRNPA1 reduces SNCA protein primarily through transcript destabilization. Unexpectedly, hnRNPA1 silencing increased total SNCA protein levels 2-fold (p < 0.05) in both cell lines (Fig. 8 c) despite reducing steady-state SNCA mRNA levels. RPL22-RiboTag analysis revealed a 50% (p < 0.05) increase in ribosomal association of long SNCA transcripts following hnRNPA1 depletion (Fig. 8 d), indicating enhanced translation efficiency. SNCA protein is degraded through two major pathways: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Within the ALP, both macroautophagy and chaperone-mediated autophagy (CMA) are implicated in SNCA degradation, with LAMP2A as the SNCA receptor in CMA [ 34 ]. To explore whether hnRNPA1 affects SNCA protein levels by interfering with these catabolic pathways, its role in both systems was investigated. The UPS activity was assessed using the fluorogenic substrate Suc-LLVY-AMC, which releases fluorescent AMC upon proteasomal cleavage. hnRNPA1 overexpression enhanced proteasomal activity by 25% (p < 0.01), while its silencing had no effect (Fig. 8 e). These findings were validated using a GFP-based reporter system that allows quantification of ubiquitin-proteasome-dependent proteolysis [ 35 ]. This system utilizes three constructs: Ub-R-GFP and Ub-GR76V-GFP, which incorporate N-end rule and ubiquitin fusion degradation signals, respectively, making them proteasome targets, and Ub-M-GFP, which upon ubiquitin cleavage remains stable like unmodified GFP. Western blot analysis showed that hnRNPA1 overexpression decreased R-GFP and GR76V-GFP levels by 50% (p 0.05), respectively, while its depletion increased these proteins by 60% (p < 0.05) and 98% (p < 0.05) (Fig. 8 f), confirming that hnRNPA1 promotes UPS activity. To assess ALP function, p62 and LAMP2 levels were monitored, and while hnRNPA1 modulation did not affect their expression (Fig. 8 g), autophagic flux was analyzed using a specialized reporter system. This system employs RlucLC3wt, which becomes degraded inside autophagosomes, and RlucLC3G120A, which cannot undergo lipidation and remains stable, serving as a control [ 36 ]. The analysis revealed that hnRNPA1 overexpression enhanced macroautophagy, while its depletion had no effect (Fig. 8 h). Collectively, these findings demonstrate that hnRNPA1 reduces SNCA protein levels through multiple mechanisms: promoting transcript destabilization and degradation via enhanced UPS and macroautophagy activities, while its depletion increases SNCA protein by enhancing the ribosomal association of long transcripts and reducing UPS activity. DISCUSSION Dysregulation of SNCA expression is a hallmark of several neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Understanding the mechanisms regulating SNCA expression is essential for therapeutic development. Previous research has established that the SNCA 5'UTR contains an internal ribosome entry site (IRES) element enabling protein synthesis during stress when most mRNAs are sequestered from translation [ 37 ]. Additionally, SNPs in the SNCA 3'UTR associated with PD [ 12 , 38 – 40 ] and altered SNCA APA patterns in PD patients [ 12 , 41 ] highlight the significance of SNCA regulatory elements in disease pathogenesis. The current study demonstrates that SNCA transcripts with longer 3'UTRs display distinct nuclear retention patterns, forming nuclear speckles and showing preferential somatic localization. The increased nuclear retention and accelerated decay of longer SNCA transcripts, together with their reduced translation efficiency, indicate that alternative polyadenylation (APA) is an effective mechanism for modulating SNCA protein levels. This regulatory layer may be particularly relevant in neurons, where precise control of local protein synthesis is essential for synaptic function and plasticity. Identifying hnRNPA1 as a regulator of SNCA expression revealed multiple mechanistic control layers (Fig. 9 ). hnRNPA1 binds directly to both proximal and distal segments of the SNCA 3'UTR and influences gene expression through distinct pathways. At the transcriptional level, hnRNPA1 enhances SNCA mRNA expression, consistent with its previously described role in transcriptional regulation of other genes [ 20 , 21 ]. The promotion of 3'UTR lengthening by hnRNPA1 appears to occur primarily through enhanced transcription elongation via P-TEFb rather than through direct interference with the core APA machinery or U1 snRNP-dependent mechanisms. This finding aligns with previous studies showing that transcription elongation rates can influence APA site selection [ 11 ], although other processes, including epigenetic regulation through chromatin and histone modifications, cannot be excluded [ 42 ]. hnRNPA1's impact on SNCA extends beyond transcription and APA (Fig. 9 ). The protein reduces the nuclear export of longer SNCA transcripts while promoting the decay of shorter variants in the cytoplasm. This differential regulation coincides with increased miR-7 expression and reduced PABPC1 levels, suggesting that compartment-specific effects contribute to transcript-specific stability. The predominant role of cytoplasmic rather than nuclear degradation is supported by preliminary observations that depletion of nuclear exosome components DIS3 and DIS3L does not affect SNCA transcript levels after hnRNPA1 overexpression (data not shown). During prolonged hnRNPA1 depletion, cells appear to adapt their RNA processing and decay machinery, reaching a new steady state with lower overall SNCA mRNA levels but enhanced protection of remaining transcripts. Such temporal dynamics may be particularly relevant in conditions where chronic alterations in RBP levels occur. At the protein level, hnRNPA1 reduces SNCA abundance through multiple mechanisms (Fig. 9 ). Beyond its effects on transcript levels and localization, hnRNPA1 enhances proteasomal activity and macroautophagy. This finding is particularly intriguing given that both the ubiquitin-proteasome system and autophagy-lysosome pathway are implicated in synucleinopathies [ 34 ]. When hnRNPA1 is depleted, increased SNCA protein levels result from enhanced ribosomal association of long transcripts and reduced proteasomal function, as demonstrated by the RiboTag and proteolysis assays. The relationship between hnRNPA1 and neurodegenerative disease extends beyond its regulation of SNCA. Reduced hnRNPA1 levels have been reported in Alzheimer's disease and ALS cases with TDP43 aggregates [ 43 , 44 ], and knockout hnRNPA1 mice exhibit cognitive dysfunction [ 43 ]. Furthermore, overexpression of hnRNPA1 has been shown to reduce the formation of TDP43 aggregates [ 45 ]. Additionally, hnRNPA1's role in telomere biogenesis and maintenance [ 46 , 47 ] connects it to cellular aging mechanisms [ 48 ], a major risk factor for neurodegenerative disorders. In the context of this work, the hematopoietic- and neurologic-expressed sequence 1 (Hn1/JPT1) protein, which mitigates senescence phenotypes, is regulated by hnRNPA1 at the APA level [ 49 ]. In contrast to SNCA mRNA, hnRNPA1 downregulation leads to 3'UTR lengthening of HN1 mRNA, resulting in decreased protein production [ 49 ], highlighting the context-dependent nature of hnRNPA1's regulatory effects. These findings open several important avenues for future research. The functional consequences of SNCA 3'UTR isoform expression need to be examined in vivo , particularly in synaptic function and neurodegeneration. Additionally, determining whether hnRNPA1 expression is dysregulated in PD could provide new insights into disease mechanisms, especially given its role in regulating genes involved in cellular senescence and its connection to PD-associated SNPs in the SNCA 3'UTR. CONCLUSIONS This study reveals that the SNCA 3'UTR governs critical regulatory events controlling alpha-synuclein expression and localization. Moreover, hnRNPA1 emerges as a central orchestrator of SNCA expression at both transcriptional and post-transcriptional levels, including APA, nuclear export, mRNA stability, and protein clearance. These insights advance our understanding of SNCA regulation and suggest that modulating hnRNPA1 or SNCA 3'UTR dynamics may provide therapeutic avenues for synucleinopathies. Abbreviations ALP: autophagy-lysosome pathway; APA: alternative polyadenylation; CMA: chaperone-mediated autophagy; DLB: dementia with Lewy bodies; LB: Lewy bodies; hnRNPA1: A/U-rich element binding factor 1; IRES: internal ribosome entry site; MSA: multiple system atrophy; LC-MS/MS: liquid chromatography (LC) with mass spectrometry (MS); ns: not significant; PD: Parkinson's disease; RBP: RNA binding protein; SNCA: alpha-synuclein; SNP: Single-nucleotide polymorphism; UPS: ubiquitin-proteasome system; UTR: untranslated region. Declarations ACKNOWLEDGMENTS Dr. G. Stoecklin (Center for Molecular Biology of Heidelberg University and Mannheim Institute for Innate Immunoscience of Heidelberg University, Germany) is acknowledged for providing the CNOT1 plasmid. FUNDING This study was supported by grants from the Michael J. Fox Foundation for Parkinson's Research (Grant ID16186), the Greek General Secretariat for Research and Innovation (Τ2EDK-01291, TAEDR-0535850), and Empirikion Foundation (2021) to E.D. AUTHOR CONTRIBUTIONS E.D. designed primers and prepared plasmids. F.K. and E.D. conducted molecular and biochemical experiments. A.A.K. and G.T.T. performed proteomics experiments. F.K. and E.D. analyzed data. F.K. created graphs and diagrams. E.D. designed and supervised experiments and wrote the manuscript. All authors reviewed and approved the final manuscript. DATA AVAILABILITY This article and the supplementary information files have included all generated data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [50] partner repository with the dataset identifier PXD060368. ETHICS APPROVAL Rodent brain tissue was obtained in accordance with the European Union (2003/65/CE) guidelines regarding the use of laboratory animals. 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Cell 184:306–322. 10.1016/j.cell.2020.12.028 Jia Q, Nie H, Yu P, Xie B, Wang C, Yang F, Wei G, Ni T (2019) HNRNPA1-mediated 3' UTR length changes of HN1 contributes to cancer- and senescence-associated phenotypes. Aging 11:4407–4437. 10.18632/aging.102060 Perez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, John NS, Prakash A, Walzer M, Wang S et al (2025) The PRIDE database at 20 years: 2025 update. Nucleic Acids Res 53:D543–D553. 10.1093/nar/gkae1011 Supplementary Files Graphicalabstract.tif Supplementarytable1.docx Supplementarytable2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6294566","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435195691,"identity":"0ee8a2b9-d2b2-475d-b2cc-5a3b60e2a9fc","order_by":0,"name":"Fedon-Giasin Kattan","email":"","orcid":"","institution":"Biomedical Research Foundation of the Academy of Athens: Idryma Iatrobiologikon Ereunon tes Akademias Athenon","correspondingAuthor":false,"prefix":"","firstName":"Fedon-Giasin","middleName":"","lastName":"Kattan","suffix":""},{"id":435195692,"identity":"da7a660b-f678-43bd-aafe-c8a486e3c6ac","order_by":1,"name":"Athanasios K Anagnostopoulos","email":"","orcid":"","institution":"Biomedical Research Foundation of the Academy of Athens: Idryma Iatrobiologikon Ereunon tes Akademias Athenon","correspondingAuthor":false,"prefix":"","firstName":"Athanasios","middleName":"K","lastName":"Anagnostopoulos","suffix":""},{"id":435195693,"identity":"fb66d3ce-c530-40d8-80d5-40f113490bb9","order_by":2,"name":"George T Tsangaris","email":"","orcid":"","institution":"Biomedical Research Foundation of the Academy of Athens: Idryma Iatrobiologikon Ereunon tes Akademias Athenon","correspondingAuthor":false,"prefix":"","firstName":"George","middleName":"T","lastName":"Tsangaris","suffix":""},{"id":435195694,"identity":"261e13db-0988-4fee-99d6-c906101e1c13","order_by":3,"name":"Epaminondas Doxakis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYFCCBCA+IMHAL8HYAOJCSKK0SM4gUQsDg8ENCJewFn725GMSP85Y2Bvfbm588KPGTrZf7IwB49c9uLVI9jxLNuy5IZG47c7BZsOeY8nGM2fnGDDLPMOtxeBGjuEDng8SCWY3EtskeBuYEzfcBmqROIBbi/2NHIODfz5I2BvPSGyT/NtQn7ifkBYDiRzDxzw3JBg3SCS2SfM2HE7cIJ1jwPgBjxaJM8+SjWXOSCTOAPrFWObYceMZt9MKDjPg0cLfnnxM8s2xOnv+2e0PH76pqZbtn5288eEPPFqwAQ6Dwzyk6WBgf8D4g0Qto2AUjIJRMKwBAD/4W+ldxKP+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1305-0739","institution":"Biomedical Research Foundation of the Academy of Athens: Idryma Iatrobiologikon Ereunon tes Akademias Athenon","correspondingAuthor":true,"prefix":"","firstName":"Epaminondas","middleName":"","lastName":"Doxakis","suffix":""}],"badges":[],"createdAt":"2025-03-24 10:57:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6294566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6294566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79860023,"identity":"ba5c8055-9f84-43d1-8cbf-a003d1cfee4a","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21520572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcripts with longer 3'UTRs exhibit distinct subcellular localization and reduced expression. a\u003c/strong\u003e RT-qPCR analysis of nuclear and cytoplasmic fractions from SK-N-SH cells showing enhanced nuclear retention of the long \u003cem\u003eSNCA\u003c/em\u003e transcript compared to total \u003cem\u003eSNCA\u003c/em\u003etranscripts. Intronic SNCA primers validated the fractionation protocol.\u003cstrong\u003e b\u003c/strong\u003eNucleus-to-cytoplasm ratio of \u003cem\u003eSNCA\u003c/em\u003e mRNA showing progressive increase in nuclear localization with 3'UTR length in differentiated CAD cells 24h after transfection with SNCA constructs containing the four main 3'UTR length variants.\u003cstrong\u003e c\u003c/strong\u003e RNA FISH analysis revealing increased nuclear speckle formation with longer 3'UTR variants in CAD cells.\u003cstrong\u003e d\u003c/strong\u003e Stability analysis showing accelerated decay of long \u003cem\u003eSNCA\u003c/em\u003e transcript compared to total \u003cem\u003eSNCA\u003c/em\u003etranscripts in SK-N-SH cells treated with actinomycin D (5 μg/ml, RNA extracted at 0, 3, 6, and 9h).\u003cstrong\u003e e\u003c/strong\u003e Western blot demonstrating an inverse correlation between SNCA 3'UTR length and protein expression in Neuro2a cells 48h post-transfection. GAPDH served as a loading control.\u003cstrong\u003e f\u003c/strong\u003e Fluorescence analysis showing decreased EGFP expression with increasing SNCA 3'UTR length in CAD cells, 24h after co-transfection with EGFP-SNCA-3'UTR hybrids and RFP control.\u003cstrong\u003e g\u003c/strong\u003e Dual-luciferase assay showing reduced luciferase expression from the 2529 nt SNCA 3'UTR reporter construct compared to the 575 nt SNCA 3'UTR reporter construct in SK-N-SH cells.\u003cstrong\u003e h\u003c/strong\u003e RPL22-RiboTag assay demonstrating decreased ribosomal association of long \u003cem\u003eSNCA\u003c/em\u003e transcripts in SK-N-SH cells expressing flagged RPL22.\u003cstrong\u003e i\u003c/strong\u003e Quantification showing increased cytoplasmic SNCA RNP speckles with longer 3'UTR variants in transfected CAD cells.\u003cstrong\u003e j\u003c/strong\u003e Analysis revealing preferential somatic localization of both mRNA and protein for longer SNCA 3'UTR variants in CAD cells.\u003cstrong\u003e \u003c/strong\u003eData represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, **p\u0026lt;0.01, ****p\u0026lt;0.0001). Values were normalized to \u003cem\u003eACTB+U6\u003c/em\u003e mRNA levels for RT-qPCR and ACTB for Western blot\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/e6db35943f3725bb947540e1.png"},{"id":79860017,"identity":"e5f182bd-5235-463f-ade4-6d47d0368d24","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11665383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 preferentially binds SNCA 3'UTR and regulates alternative polyadenylation. a\u003c/strong\u003e Schematic showing the workflow for identifying SNCA 3'UTR\u003csub\u003e2529\u003c/sub\u003e-interacting proteins from postnatal day 3 mouse brain lysates using biotinylated RNA pull-down and LC-MS/MS analysis. \u003cstrong\u003eb\u003c/strong\u003e Gene Ontology analysis of proteins specifically enriched on SNCA 3'UTR beads versus control beads revealing enrichment of nucleic acid binding and RNA binding molecular functions among SNCA 3'UTR-bound proteins.\u003cstrong\u003e c\u003c/strong\u003e Analysis of Biological Processes showing enrichment in mRNA processing and RNA splicing.\u003cstrong\u003e d\u003c/strong\u003e List of RBPs identified to interact with SNCA 3'UTR\u003csub\u003e2529\u003c/sub\u003e, including those that may also bind to control beads.\u003cstrong\u003e e\u003c/strong\u003e RT-qPCR analysis showing that hnRNPA1 overexpression most potently increased the ratio of long to total \u003cem\u003eSNCA\u003c/em\u003e transcripts among 17 tested RBPs in HEK293A cells 48h post-transfection.\u003cstrong\u003e f\u003c/strong\u003e ENCORI platform analysis demonstrating strong anticorrelation between hnRNPA1 and \u003cem\u003eSNCA\u003c/em\u003e mRNA levels in lower-grade brain gliomas.\u003cstrong\u003e g\u003c/strong\u003e RNA immunoprecipitation showing six-fold enrichment of endogenous \u003cem\u003eSNCA\u003c/em\u003e mRNA in hnRNPA1 complexes compared to IgG control in SK-N-SH cells.\u003cstrong\u003e h\u003c/strong\u003e Western blot analysis revealing exclusive binding of hnRNPA1 to both proximal and distal portions of SNCA 3'UTR in SK-N-SH cell lysates using biotinylated RNA fragments spanning different SNCA regions.\u003cstrong\u003e \u003c/strong\u003eData represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, **p\u0026lt;0.01). Values were normalized to \u003cem\u003eACTB+U6\u003c/em\u003e mRNA levels for RT-qPCR and ACTB for Western blot. Some elements in this image were obtained from Servier Medical Art (http://smart.servier.com/), which is permissible to use under a Creative Commons Attribution 3.0 Unported License\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/efb4ba308ca1bf52f763861c.png"},{"id":79860430,"identity":"5f4d225c-ea08-4cd3-a70b-6777aa6b1e6a","added_by":"auto","created_at":"2025-04-03 17:00:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1354601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 enhances \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etranscription. a, b\u003c/strong\u003e RT-qPCR analysis of nuclear RNA 48h after hnRNPA1 modulation in SK-N-SH cells showing significantly increased \u003cem\u003eSNCA\u003c/em\u003e pre-mRNA levels at intron3-exon4 \u003cstrong\u003e(a) \u003c/strong\u003eand intron5-exon6 \u003cstrong\u003e(b) \u003c/strong\u003ejunctions following hnRNPA1 overexpression, while hnRNPA1 silencing showed minimal effect. \u003cstrong\u003ec\u003c/strong\u003eAnalysis of proximal exon 6 revealing increased total \u003cem\u003eSNCA\u003c/em\u003e mRNA levels upon hnRNPA1 overexpression, with negligible impact of hnRNPA1 silencing. Data represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, ns: not significant). Values were normalized to \u003cem\u003eACTB+U6\u003c/em\u003e mRNA levels\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/39fb0ccab38ad1ead638bc1b.png"},{"id":79860018,"identity":"f16ba6dd-77f8-402e-847a-dfddce12190a","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6604755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 regulates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etranscript distribution and axonal localization. a, b\u003c/strong\u003e RT-qPCR analysis of nuclear and cytoplasmic fractions from SK-N-SH cells 48h after hnRNPA1 modulation showing \u003cstrong\u003e(a)\u003c/strong\u003eunaltered nuclear export of total \u003cem\u003eSNCA\u003c/em\u003e mRNA and \u003cstrong\u003e(b)\u003c/strong\u003e reduced cytoplasmic levels of long \u003cem\u003eSNCA\u003c/em\u003e transcripts upon hnRNPA1 overexpression with enhanced export following hnRNPA1 silencing. \u003cstrong\u003ec-e\u003c/strong\u003e RNA FISH analysis in differentiated CAD cells 24h after hnRNPA1 co-expression revealing \u003cstrong\u003e(c)\u003c/strong\u003eincreased nuclear speckles for the shortest SNCA transcript (290 nt), \u003cstrong\u003e(d)\u003c/strong\u003eenhanced cytoplasmic speckle formation for shorter transcripts (290 and 575 nt), and \u003cstrong\u003e(e)\u003c/strong\u003e reduced axonal localization of longer transcripts (1075-2529 nt). \u003cstrong\u003ef\u003c/strong\u003e Immunofluorescence analysis 24h after co-expression revealing corresponding decreases in axonal SNCA protein levels for longer 3'UTR variants in the presence of hnRNPA1. Data represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Values were normalized to \u003cem\u003eACTB+U\u003c/em\u003e6 mRNA levels for RT-qPCR and ACTB for immunofluorescence quantification\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/5bba94715053fbcbbde9d7d0.png"},{"id":79860022,"identity":"d694dcc3-dfa0-46a7-bc91-16590d484d04","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13193239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 modulation affects steady-state levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etranscripts. a-d\u003c/strong\u003e RT-qPCR analysis 48h after hnRNPA1 overexpression in SK-N-SH and HEK293A cells showing \u003cstrong\u003e(a)\u003c/strong\u003e robust hnRNPA1 expression, \u003cstrong\u003e(b)\u003c/strong\u003e decreased total \u003cem\u003eSNCA\u003c/em\u003e mRNA levels, (\u003cstrong\u003ec\u003c/strong\u003e) increased expression of the long \u003cem\u003eSNCA\u003c/em\u003e transcript, and \u003cstrong\u003e(d)\u003c/strong\u003eelevated ratio of long-to-total \u003cem\u003eSNCA\u003c/em\u003e transcripts. \u003cstrong\u003ee-h\u003c/strong\u003e RT-qPCR analysis 48h after hnRNPA1 shRNA transfection in both cell lines demonstrating \u003cstrong\u003e(e)\u003c/strong\u003eeffective hnRNPA1 silencing, \u003cstrong\u003e(f)\u003c/strong\u003e reduced total \u003cem\u003eSNCA\u003c/em\u003e mRNA levels, \u003cstrong\u003e(g)\u003c/strong\u003edecreased expression of the long transcript, and \u003cstrong\u003e(h)\u003c/strong\u003e unchanged ratio of long-to-total \u003cem\u003eSNCA\u003c/em\u003e transcripts. Data represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Values were normalized to \u003cem\u003eACTB+U6\u003c/em\u003e mRNA levels\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/1932f8be92f300227e0f8495.png"},{"id":79860027,"identity":"098a0bb9-3bd6-46e5-88ff-bb8c2fdc1492","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10668563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 regulates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA stability, PABPC1 protein levels, and miRNA pathways. a, b\u003c/strong\u003e Stability analysis of SK-N-SH cells 48h after hnRNPA1 overexpression showing \u003cstrong\u003e(a)\u003c/strong\u003e decreased total \u003cem\u003eSNCA\u003c/em\u003e mRNA stability and \u003cstrong\u003e(b)\u003c/strong\u003e unaltered stability of the long transcript (3'UTR\u003csub\u003e2529\u003c/sub\u003e) following actinomycin D treatment. \u003cstrong\u003ec, d\u003c/strong\u003e Similar stability analysis 48h after hnRNPA1 silencing revealing \u003cstrong\u003e(c)\u003c/strong\u003e increased total \u003cem\u003eSNCA\u003c/em\u003e mRNA stability and \u003cstrong\u003e(d)\u003c/strong\u003e unchanged stability of the long transcript following actinomycin D treatment.\u003cstrong\u003e e\u003c/strong\u003e Western blot analysis and quantification showing decreased PABPC1 protein levels upon hnRNPA1 overexpression in SK-N-SH cells, with no effect after hnRNPA1 silencing.\u003cstrong\u003e f, g\u003c/strong\u003e RT-qPCR analysis in SK-N-SH cells 48h after hnRNPA1 modulation showing \u003cstrong\u003e(f)\u003c/strong\u003e increased mature miR-7 levels with hnRNPA1 overexpression, while \u003cstrong\u003e(g)\u003c/strong\u003e miR-153 expression remained unaffected.\u003cstrong\u003e h, i\u003c/strong\u003e RT-qPCR analysis in SK-N-SH cells 48h after co-transfection with miR-7/153 showing decreased \u003cstrong\u003e(h)\u003c/strong\u003e total SNCA and \u003cstrong\u003e(i)\u003c/strong\u003e long \u003cem\u003eSNCA\u003c/em\u003e transcript levels, with similar effects regardless of hnRNPA1 modulation.\u003cstrong\u003e \u003c/strong\u003eData represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, **p\u0026lt;0.01). Values were normalized to \u003cem\u003eACTB+U6\u003c/em\u003e mRNA levels for RT-qPCR and ACTB for Western blot\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/84d27b7a16c7d70b812340c0.png"},{"id":79860862,"identity":"245b2ce9-c527-4977-bd87-752dec58534f","added_by":"auto","created_at":"2025-04-03 17:08:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":15187550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 binding to SNCA 3'UTR influences transcription elongation but not core polyadenylation machinery. a \u003c/strong\u003eSchematic representation of the polyadenylation machinery showing the interaction of core complexes (CPSF, CSTF, CFIm) with specific RNA sequence elements.\u003cstrong\u003e b \u003c/strong\u003eWestern blot analysis of biotinylated SNCA 3'UTR\u003csub\u003e2529\u003c/sub\u003e pull-down from SK-N-SH cell lysates 48h after hnRNPA1 modulation showing unchanged binding of core polyadenylation factors (NUDT21, CPSF6, CPSF4, WDR33, CSTF64) despite increased hnRNPA1 association.\u003cstrong\u003e c\u003c/strong\u003e Analysis of SNCA intronic expression in SK-N-SH cells 48h after hnRNPA1 transfection following DRB treatment for 2 h and release. Control cells showed an expected progressive increase in \u003cem\u003eSNCA\u003c/em\u003e transcripts, while hnRNPA1-expressing cells demonstrated resistance to DRB, suggesting maintained P-TEFb engagement with RNA Pol II. Data represent mean ± SD from at least 3 independent experiments. Input samples represent 10% of the total lysate used for pull-down\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/9c7de92e64131032bcedd660.png"},{"id":79860031,"identity":"b3acdcc7-4cb2-44a8-81a1-dd63b6c22224","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9371337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 regulates SNCA protein levels through multiple pathways. a \u003c/strong\u003eWestern blot analysis and quantification in SK-N-SH and HEK293A cells 48h after hnRNPA1 overexpression showing decreased SNCA protein levels.\u003cstrong\u003e b\u003c/strong\u003e RPL22-RiboTag analysis in SK-N-SH cells showing unaltered association of total \u003cem\u003eSNCA\u003c/em\u003e transcripts with ribosomes upon hnRNPA1 overexpression.\u003cstrong\u003e c\u003c/strong\u003e Western blot analysis and quantification 48h after hnRNPA1 silencing revealing increased SNCA protein levels in both cell lines.\u003cstrong\u003e d\u003c/strong\u003e RPL22-RiboTag analysis showing enhanced ribosomal association of long \u003cem\u003eSNCA\u003c/em\u003e transcripts following hnRNPA1 depletion in SK-N-SH cells.\u003cstrong\u003ee \u003c/strong\u003eProteasomal activity assay using Suc-LLVY-AMC showing increased activity with hnRNPA1 overexpression.\u003cstrong\u003e f\u003c/strong\u003e Western blot analysis and quantification of GFP-based UPS reporters 48h after hnRNPA1 modulation demonstrating enhanced proteasomal degradation with hnRNPA1 overexpression and reduced activity upon its silencing.\u003cstrong\u003e g\u003c/strong\u003e Western blot analysis showing unaltered p62 and LAMP2 levels 48h after hnRNPA1 modulation.\u003cstrong\u003e h\u003c/strong\u003e Analysis of autophagic flux using RlucLC3 reporters revealing enhanced macroautophagy with hnRNPA1 overexpression. Data represent mean ± SD from at least 3 independent experiments (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Values were normalized to \u003cem\u003eACTB+U6\u003c/em\u003e mRNA levels for RT-qPCR and ACTB for Western blot\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/4564b068d0ef0d27c3a03ef0.png"},{"id":79860045,"identity":"4f57a649-a129-4cac-8a4e-536cb03282f7","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":10292231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehnRNPA1 orchestrates multi-level regulation of SNCA expression.\u003c/strong\u003e In the nucleus, hnRNPA1 exerts dual effects: enhancing \u003cem\u003eSNCA\u003c/em\u003e transcription while promoting 3'UTR lengthening by sequestering 7SK RNA, a repressor of the transcription elongation factor P-TEFb. Additionally, hnRNPA1 reduces the nuclear export of longer \u003cem\u003eSNCA\u003c/em\u003etranscripts, protecting them from cytoplasmic degradation mechanisms. In the cytoplasm, hnRNPA1 promotes the decay of shorter transcripts through increased miR-7 levels and reduced PABPC1 expression. It also activates proteasome-mediated degradation and enhances autophagic flux, decreasing SNCA protein levels. In the axonal compartment, HNRNPA1 reduces axonal transport of longer \u003cem\u003eSNCA\u003c/em\u003e transcripts and their translation. The integrated actions of hnRNPA1 result in tightly controlled SNCA expression through coordinated regulation of transcription, RNA processing, stability, localization, and protein turnover. Solid arrows indicate direct effects and dashed arrows represent indirect or sequential effects\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/2f8580bb61b33ef3dbec19fc.png"},{"id":79860431,"identity":"5ba0ba45-d155-4458-863d-0c53ad0522c4","added_by":"auto","created_at":"2025-04-03 17:00:28","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4440772,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/818d5b4101848c7db2df5112.tif"},{"id":79860015,"identity":"cb21d437-1fa6-420f-acad-dae670bbce21","added_by":"auto","created_at":"2025-04-03 16:52:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19508,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/9b3b44ecb7293918290e7a82.docx"},{"id":79860861,"identity":"72a05efa-abaa-4e0a-9e6c-6b40d1a0cdf5","added_by":"auto","created_at":"2025-04-03 17:08:28","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":143657,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6294566/v1/fc2f579b0fa2bb94405c8db4.xlsx"}],"financialInterests":"","formattedTitle":"hnRNPA1 orchestrates multi-layered regulation of SNCA expression","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSNCA is an abundant presynaptic protein that acts as a chaperone for SNARE-complex assembly, regulating synaptic neurotransmission. Accumulation of SNCA has been implicated in the development of Parkinson's disease, multiple system atrophy (MSA), and Lewy body dementia (LBD), collectively known as alpha-synucleinopathies. The underlying causes of SNCA-induced neurodegeneration are multifaceted, with misfolded and aggregated forms released from neurons facilitating the spread of pathology in a prion-like manner (reviewed by [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]). Maintaining physiological levels of SNCA expression is thus essential for neuronal function and viability, yet the molecular mechanisms controlling SNCA expression levels in neurons remain incompletely understood.\u003c/p\u003e \u003cp\u003ePost-transcriptional regulation of gene expression is particularly crucial in neurons, given their complex architecture and requirement for local protein synthesis. In eukaryotes, mature mRNA has a tripartite structure consisting of a 5'UTR, a protein-coding region, and a 3'UTR. While the 5'UTR primarily governs translation initiation, the 3'UTR is central to mRNA subcellular localization, stability, translation regulation, and termination. Notably, the mRNAs of presynaptic genes -including \u003cem\u003eSNCA\u003c/em\u003e- possess significantly longer 3'UTRs than other transcripts, suggesting an expanded scope for post-transcriptional regulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRNA-binding proteins (RBPs) tightly control these processes by binding to specific cis-elements along the RNA sequence. Given the architectural complexity of neurons, characterized by small soma and extensive projections, RBPs play a vital role in the nervous system, orchestrating neurogenesis, neurite outgrowth, synapse formation, and plasticity (reviewed in [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]). Among the various RBPs, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is an evolutionarily conserved, highly expressed factor implicated in diverse RNA processing events, including transcription, telomere maintenance, splicing, miRNA maturation, mRNA stability, and translation (reviewed in [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]). hnRNPA1 possesses two globular RNA recognition motifs for sequence-specific RNA binding and an unstructured low-complexity C-terminal domain, mediating protein-protein interactions involved in stress granule assembly [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This low-complexity domain also exhibits prion-like properties, leading to amyloid aggregation of hnRNPA1, which has been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP), especially when mutations are inherited [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. hnRNPA1 recognizes a variety of RNA sequences with specificity towards a 5'-YAG-3' motif (where Y is C or U) (reviewed in [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eAPA-mediated lengthening of 3\u0026prime; UTRs is particularly prevalent in neurons, especially in presynaptic mRNAs like SNCA. This process constitutes a distinct layer of gene regulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and is considered necessary for the spatial organization of protein synthesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Human \u003cem\u003eSNCA\u003c/em\u003e mRNA undergoes alternative cleavage and polyadenylation, generating 3'UTR variants of approximately 290, 575, 1075, and 2,529 nucleotides (nt). The 575 and 2,529 nt forms constitute roughly 40% and 35% of SNCA transcripts in the human cortex, respectively [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The longer isoform is upregulated during differentiation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and linked to PD pathology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], suggesting its involvement in disease mechanisms.\u003c/p\u003e \u003cp\u003eThis study investigates how different SNCA 3'UTR isoforms influence protein expression and localization in neurons, focusing on their role in maintaining appropriate SNCA levels. Through an unbiased proteomic approach, hnRNPA1 emerged as a key regulator of SNCA expression via multiple mechanisms. These results provide new insights into the post-transcriptional control of SNCA and highlight potential therapeutic targets for modulating SNCA expression in neurodegenerative diseases.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eFor Western blot analysis, the following primary antibodies were used: rabbit polyclonal anti-hnRNPA1 (1:2000, sc-32301, Santa Cruz, CA, USA), mouse monoclonal anti-SNCA (1:1000, sc-7011, Santa Cruz and 1:1000, 610787, BD Transduction), rabbit polyclonal anti-CNOT1 (1:1000, 14276-1-AP, Proteintech Europe, Manchester, UK), rabbit polyclonal anti-CNOT7 (1:1000, 14102-1-AP, Proteintech), rabbit polyclonal anti-PABP1 (1:1000, 10970-1-AP, Proteintech), mouse monoclonal anti-CNOT6 (1:1000, sc-81231, Santa Cruz), mouse monoclonal anti-LAMP2A (1:1000, ab125068, Abcam), mouse anti-GAPDH-HRP conjugated (1:5000, HRP-60004, Proteintech), and anti-puromycin (1:3000, MABE343, Merck Millipore, Darmstadt, Germany). Mouse monoclonal anti-SNCA (1:250, 610787, BD Transduction) and mouse monoclonal anti-FLAG (1:500, F1804, Sigma-Aldrich) were used for immunofluorescence and RNA FISH experiments. Secondary antibodies for Western blot included HRP-conjugated mouse (1:5000, #7076, Cell Signaling Technologies, Danvers, MA, USA) and rabbit (1:5000, #7074, Cell Signaling) antibodies. For immunofluorescence, goat anti-mouse Alexa Fluor 488 (1:500, A11029, Invitrogen) and goat anti-rabbit Alexa Fluor 568 (1:500; A11036, Invitrogen) were used. Immunoprecipitation control antibodies included normal mouse IgG (2 \u0026micro;g, sc-2025, Santa Cruz).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of DNA constructs\u003c/h3\u003e\n\u003cp\u003eAll primers are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The human hnRNPA1 CDS, SNCA CDS\u0026thinsp;+\u0026thinsp;3'UTRs, and SNCA 3'UTRs were PCR-amplified using Phusion polymerase (ThermoFisher) from SK-N-SH cDNA. Plasmid construction was performed as follows: hnRNPA1 CDS (KpnI/NotI sites in pENTR-GD), SNCA CDS\u0026thinsp;+\u0026thinsp;3'UTRs (BamHI/HindIII sites in paavCAG-pre-mGRASP-mCerulean-2A-nls-mCherry) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], SNCA 3'UTRs for hybrid constructs (PstI/SmaI sites in EGFP-SNCA-WT) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and SNCA 3'UTRs for luciferase (XhoI/NotI sites in psiCHECK-2, Promega, Madison, USA). Two hnRNPA1 shRNA plasmids targeting all \u003cem\u003ehnRNPA1\u003c/em\u003e transcripts were generated using Block-iT U6 RNAi Entry vector Kit (ThermoFisher). All constructs were sequence-verified by Sanger sequencing (CeMIA SA, Larisa, Greece).\u003c/p\u003e\n\u003ch3\u003eCell culture and transfection\u003c/h3\u003e\n\u003cp\u003eSK-N-SH, HEK293A, and Neuro2A cells were maintained in high-glucose DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich). CAD cells were cultured in DMEM/F12 (Sigma-Aldrich) supplemented with 8% FBS. For differentiation experiments, CAD cells were maintained in a medium containing 2% FBS post-transfection. All cells were grown at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e (ThermoForma, ThermoFisher).\u003c/p\u003e \u003cp\u003eTransfections were performed at plating using jetOPTIMUS reagent according to the manufacturer's instructions (Polyplus, Illkirch, France). A 1:1 ratio of DNA (\u0026micro;g) to jetOPTIMUS reagent (\u0026micro;l) was used for plasmid transfections. Cells were transfected with 100 nM siRNA for siRNA experiments using jetPRIME reagent. Transfection efficiency was monitored using EmGFP plasmid, achieving approximately 80% efficiency in SK-N-SH, HEK293A, and Neuro2A cells at 48 h post-transfection. Cells were harvested 24\u0026ndash;48 h post-transfection for analysis.\u003c/p\u003e\n\u003ch3\u003eTotal RNA extraction, cDNA synthesis, and PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from SK-N-SH cells using TRI Reagent (Molecular Research Centre Inc, Cincinnati, OH, USA). RNA quantity and quality were verified spectrophotometrically (A260/280\u0026thinsp;\u0026gt;\u0026thinsp;1.9). For standard cDNA synthesis, RNA was reverse-transcribed with M-MLV reverse transcriptase (ThermoFisher) and random hexamers. For miRNA detection, RNA was polyadenylated using poly(A) polymerase (NEB) before reverse transcription, as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The resulting cDNA was diluted 11-fold and stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eQuantitative PCR was performed using Kapa SYBR Fast Universal 2\u0026times; qPCR Master Mix (Kapa Biosystems, Roche, Basel, Switzerland) in 96-well PCR microplates (Azenta, Burlington, MA, USA) on a CFX OPUS real-time PCR system (BioRad, Richmond, CA, USA). Negative RT controls were included, and all samples were analyzed in technical triplicates. Data were evaluated using the 2^\u0026minus;ΔΔCT method, normalizing to GAPDH and U6 mRNA levels. Primer sequences are in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003emRNA half-life measurement\u003c/h3\u003e\n\u003cp\u003eFor mRNA stability, SK-N-SH cells were transfected with hnRNPA1 or control plasmids for 36\u0026ndash;48h before treatment with actinomycin D (5 \u0026micro;g/ml; MedChemExpress, Monmouth Junction, NJ, USA). Cells were harvested at 0, 3, 6, and 9h post-treatment, and RNA was analyzed by RT-qPCR. One-phase decay non-linear regression was done in GraphPad Prism (Y0\u0026thinsp;=\u0026thinsp;100).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNucleocytosolic fractionation\u003c/h2\u003e \u003cp\u003eCell fractionation was performed using ice-cold HLB buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EGTA, 0.1% NP40) supplemented with 40U/mL RNAseOUT. After 10 min on ice, lysates were centrifuged at 800\u0026times;g for 3 min at 4\u0026deg;C to separate cytoplasmic (supernatant) and nuclear (pellet) fractions. Nuclear pellets were washed three times with HLB buffer before resuspension in TRI Reagent. During RNA extraction, nuclear fractions underwent an additional 65\u0026deg;C incubation for 10 min during phase separation to facilitate the release of membrane-bound mRNAs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRPL22-RiboTag assay\u003c/h3\u003e\n\u003cp\u003eFor each condition, 1\u0026times;10⁶ SK-N-SH cells were transfected with RPL22-FLAG alone or together with hnRNPA1 or control plasmids. After 48 h, cells were washed twice with ice-cold PBS and harvested in PLB lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% NP-40, 1 mM DTT) supplemented with 1\u0026times; cOmplete protease inhibitor cocktail and 40U/mL RNAseOUT. After 30-min incubation on ice, lysates were cleared by centrifugation (16,000\u0026times;g, 10 min, 4\u0026deg;C).\u003c/p\u003e \u003cp\u003ePrior to immunoprecipitation, 10% of the supernatant was reserved as input control. FLAG G1 resin (MedChemExpress) was prepared by washing three times with TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). The washed resin was resuspended in TBS containing 40U/mL RNAseOUT and 1\u0026times; protease inhibitor cocktail. Cleared lysates were combined with prepared resin and incubated for 4 h at 4\u0026deg;C on a rotating mixer. Following incubation, beads were washed three times with NT2 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.05% NP-40). RNA was extracted from both beads and input samples using TRI Reagent.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn situ\u003c/b\u003e \u003cb\u003ehybridization\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eSNCA\u003c/em\u003e mRNA was detected using Stellaris\u0026reg; RNA FISH probes (LGC Biosearch Technologies, Hoddesdon, UK). Differentiated CAD cells on poly-D-lysine-coated glass coverslips were washed once with PBS, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed twice with PBS, and permeabilized in 70% ethanol at 4\u0026deg;C for 24h. Following aspiration of ethanol, cells were washed once with Stellaris wash solution A and incubated for 8 h at 37\u0026deg;C with Stellaris hybridization solution containing custom-designed SNCA 3'UTR probes (set of 30 sequences spanning the length of the 3'UTR, conjugated with Quasar\u0026trade; 570nm fluorophore; 1:100 dilution) and primary anti-synuclein antibody. After hybridization, cells were washed twice with Stellaris wash solution A and incubated with Alexa Fluor\u0026trade; 488nm secondary antibody (1:500) in wash solution A for 1 h at room temperature. Nuclear staining was performed using DAPI (1 \u0026micro;g/mL) for 3 min, followed by a final wash with Stellaris wash solution B. Coverslips were mounted on microscope slides using SlowFade Gold antifade reagent (Invitrogen) and sealed with nail polish. Images were acquired using a Leica SP5 confocal microscope with a 63\u0026times; oil immersion objective, maintaining consistent acquisition parameters across all samples. Z-stacks were collected at 0.4 \u0026micro;m intervals, covering the entire cell depth.\u003c/p\u003e\n\u003ch3\u003eAffinity pull-down of biotinylated RNA\u003c/h3\u003e\n\u003cp\u003eSNCA mRNA domains (5'UTR, CDS, 3'UTR\u003csub\u003e575\u003c/sub\u003e, and 3'UTR\u003csub\u003e2529\u003c/sub\u003e) were PCR-amplified from SK-N-SH cDNA using domain-specific primers containing the T7 RNA polymerase promoter sequence (5'-AGTAATACACTCACTATAGGG-3') (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e transcription was performed in reactions containing 0.75 \u0026micro;g PCR product, 1\u0026times; T7 buffer, 50 mM DTT, 40U/mL RNAseOUT, NTPs (20 mM A/U/G, 16.3 mM C, 3.7 mM biotin-11-CTP; Roche), and T7 polymerase (Takara Bio) at 42\u0026deg;C for 2 h. Template DNA was removed using RNase-free DNAase I (NEB) for 20 min at 37\u0026deg;C, followed by LiCl precipitation of RNA. RNA probes were prepared in structure buffer (10 mM Tris pH 7.0, 0.1 M KCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e), heated to 90\u0026deg;C for 2 min, cooled on ice for 2 min, and equilibrated at room temperature for 20\u0026ndash;30 min for proper folding.\u003c/p\u003e \u003cp\u003eOne postnatal day 3 mouse brain or 10\u003csup\u003e7\u003c/sup\u003e SK-N-SH cells were used for each pull-down. Cell lysates were prepared in ice-cold NT2 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.05% NP-40) supplemented with protease inhibitors. Following a 45-min rotation at 4\u0026deg;C, lysates were centrifuged at 16,000\u0026times;g for 10 min at 4\u0026deg;C. RNA-protein binding was performed by incubating lysates with 2 \u0026micro;g of RNA probes for 2 h at room temperature, followed by adding pre-washed streptavidin magnetic beads for another 2 h. After three NT2 buffer washes, bound proteins were eluted using NT2 buffer containing 1\u0026times; Laemmli buffer (250 mM Tris-HCl pH 6.8, 6% SDS, 30% β-mercaptoethanol, 40% glycerol, 0.005% bromophenol blue) at room temperature for 15 min followed by 10-min boiling.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePeptide Generation and 1-D nanoLC-MS/MS analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProtein extraction and peptide generation were performed according to established protocols [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, proteins were extracted in 7 M urea buffer containing 80 mM triethyl ammonium bicarbonate (TEAB), followed by 30-min sonication in a water bath. Reduction and alkylation were performed using 10 mM dithiothreitol and 55 mM iodoacetamide, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eLC-MS/MS analysis was performed using an LTQ Orbitrap Elite mass spectrometer interfaced with a Dionex Ultimate 3000 HPLC system (Thermo Scientific, Rockford, IL, USA) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Peptide separation was achieved using two Thermo Scientific columns in series: a PepMap RSLC C18 column (100 \u0026Aring;, 3-\u0026micro;m-bead-packed 15-cm column) followed by a PepMap RSLC C18 column (100 \u0026Aring;, 2-\u0026micro;m-bead-packed 50-cm column). Peptides were eluted at a flow rate of 3 nL min-1 using mobile phase A (0.1% formic acid in water) and mobile phase B (99% acetonitrile, 0.1% formic acid). The gradient program was optimized: 2.0% B for 10 min, linear increase from 2.0\u0026ndash;35.0% B over 325 min, increase to 80.0% B and hold for 10 min, return to 2.0% B, and re-equilibrate for 10 min.\u003c/p\u003e \u003cp\u003eMass spectrometric data were acquired using a data-dependent acquisition strategy. Full MS scans were acquired in the Orbitrap at a resolution of 60,000 (at m/z 400) with a scan range of 250\u0026ndash;1250 m/z and a maximum injection time of 250 msec. The top 20 most intense precursor ions from each MS scan were selected for fragmentation using higher energy collision dissociation (HCD) with a normalized collision energy of 36%. MS/MS spectra were acquired in the Orbitrap at a resolution of 15,000 with a maximum injection time of 120 msec. Dynamic exclusion parameters included one repeat count, 30-sec repeat duration, 120-sec exclusion duration, and a\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 m/z low and \u0026plusmn;\u0026thinsp;1.6 m/z high exclusion mass width. Lock mass calibration was performed using m/z 445.120025 as an internal standard.\u003c/p\u003e \u003cp\u003eRaw data files were processed using the SEQUEST algorithm within Proteome Discoverer\u0026trade;. MS/MS spectra were searched against the appropriate protein database using the following parameters: parent ion mass tolerance of 20 ppm, fragment mass tolerance of 0.05 Da, trypsin as the cleavage enzyme allowing up to 2 missed cleavages, cysteine methylthio as a fixed modification, and methionine oxidation as a variable modification. Peptide identifications were filtered to 1% false discovery rate (q-value\u0026thinsp;\u0026le;\u0026thinsp;0.01) using the percolator algorithm with Delta Cn maximum set at 0.05. The minimum peptide length was set to six amino acids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation of RNP complexes (RIP)\u003c/h2\u003e \u003cp\u003eFor RNA-protein complex identification, protein A/G Sepharose beads (sc-2003, Santa Cruz Biotechnology) were pre-coated with 2 \u0026micro;g of anti-hnRNPA1 or anti-IgG antibody overnight at 4\u0026deg;C in NT2 buffer containing 5% BSA under constant agitation. Beads were washed three times with NT2 buffer. Cell extracts were prepared from 10\u003csup\u003e7\u003c/sup\u003e SK-N-SH cells using ice-cold PLB lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% NP-40, 1 mM DTT) supplemented with 1\u0026times; cOmplete protease inhibitor cocktail and 40U/mL RNAseOUT. Following 30-min incubation at 4\u0026deg;C, debris was removed by centrifugation at 16,000\u0026times;g for 10 min. Cell extracts were combined with antibody-coated beads and incubated at 4\u0026deg;C for 4 h on a rotating mixer, with a small aliquot reserved as input control. After three NT2 buffer washes, proteins were digested using proteinase K, followed by RNA extraction using Tri Reagent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of whole protein extracts and Western blotting\u003c/h2\u003e \u003cp\u003eWhole-cell lysates were prepared using ice-cold RIPA buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM EDTA, 1% Triton X-100, 0.16% Sodium Deoxycholate, 0.16% SDS) supplemented with 1\u0026times; cOmplete protease inhibitor cocktail. Following 30-min incubation on ice, lysates were centrifuged at 16,000\u0026times;g for 30 min at 4\u0026deg;C. Supernatants were stored at -80\u0026deg;C until use. Protein concentration was determined using the Bradford assay according to the manufacturer's protocol (BioRad).\u003c/p\u003e \u003cp\u003eFor immunoblotting, equal amounts of protein extracts were combined with 6\u0026times; SDS sample buffer (375 mM Tris pH 6.8, 10% SDS, 50% glycerol, 10% β-mercaptoethanol, 0.03% bromophenol blue), heated at 100\u0026deg;C for 5 min, and separated by 12% or 15% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose membranes (Amersham/Merck) and blocked with 5% non-fat milk in TBS-T (TBS containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were diluted in TBS-T and incubated overnight at 4\u0026deg;C, followed by HRP-conjugated secondary antibodies (1 h, room temperature). Immunoreactive bands were visualized using Clarity or Clarity Max ECL reagents (BioRad) and imaged using a Fusion FX6 system (Vilber, Marne-la-Vall\u0026eacute;e, France). Densitometric analysis was performed using Fiji software (NIH), with ACTD or GAPDH as the normalization controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSurface sensing of translation (SUnSET) method\u003c/h2\u003e \u003cp\u003eProtein synthesis rates were analyzed using the SUnSET method, as described by Schmidt et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. At 48 h post-transfection, SK-N-SH cells were treated with 1 \u0026micro;M puromycin (P8833, Sigma-Aldrich) for 30 min. Cells were harvested in ice-cold RIPA buffer, and protein extracts were processed for Western blotting as described above. Puromycin-labeled peptides were detected using an anti-puromycin antibody (1:3000, MABE343, Merck Millipore), followed by HRP-conjugated secondary antibody and ECL detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eLuciferase activities were measured 48 h post-transfection using the Dual-Luciferase Assay System (Promega) according to the manufacturer's protocol. Measurements were performed in a Lumat LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). Renilla luciferase (target) expression was normalized to Firefly luciferase (internal reference control), with results presented as a percent ratio of Renilla to Firefly activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGene Ontology Analysis\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) analyses for 'Molecular function' and 'Biological process' were conducted using the database for annotation, visualization, and integrated discovery (DAVID) with default parameters [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed with at least three biological replicates, and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance between two groups was determined using two-tailed Student's t-test. Multiple comparisons were analyzed using one-way ANOVA followed by Dunnett's post hoc test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were performed using GraphPad Prism version 8.0.0 (San Diego, California, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDifferential nuclear retention and distribution of SNCA 3'UTR variants\u003c/h2\u003e \u003cp\u003eThe relative abundance of the longest \u003cem\u003eSNCA\u003c/em\u003e transcript variant was approximated in SK-N-SH neuroblastoma and HEK293A cells, which express high levels of endogenous SNCA. RT-qPCR analysis using primers spanning the first segment of SNCA 3'UTR (detecting all isoforms) and the distal region of the longest \u003cem\u003eSNCA\u003c/em\u003e transcript (3'UTR\u003csub\u003e2529\u003c/sub\u003e) revealed that this extended variant comprises roughly 30% of total \u003cem\u003eSNCA\u003c/em\u003e mRNAs as reported in the human cortex, based on a 2 Ct value difference between isoforms.\u003c/p\u003e \u003cp\u003eAnalysis of nuclear and cytosolic fractions from SK-N-SH cells demonstrated distinct compartmentalization patterns among \u003cem\u003eSNCA\u003c/em\u003e variants. The fractionation protocol efficacy was validated using intronic SNCA primers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Quantification using SNCA 3'UTR proximal and distal primers revealed a four-fold higher nuclear abundance of the long transcript compared to total transcripts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating extended nuclear retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). These findings were corroborated using CNS catecholaminergic CAD cells, which were selected for their lack of endogenous SNCA expression. Following transfection with SNCA constructs containing the four main 3'UTR length variants, RNA FISH analysis demonstrated enhanced nuclear localization of 3'UTR\u003csub\u003e2529\u003c/sub\u003e transcripts, with longer variants (3'UTR\u003csub\u003e1075\u003c/sub\u003e or 3'UTR\u003csub\u003e2529\u003c/sub\u003e) forming twice as many nuclear speckles compared to shorter variants (3'UTR\u003csub\u003e290\u003c/sub\u003e or 3'UTR\u003csub\u003e575\u003c/sub\u003e) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). These findings establish distinct nuclear retention patterns among \u003cem\u003eSNCA\u003c/em\u003e variants, with longer transcripts showing enhanced nuclear accumulation and speckle formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLonger SNCA 3'UTR variants show reduced stability and translation efficiency\u003c/h2\u003e \u003cp\u003eTranscript stability analysis in actinomycin D-treated SK-N-SH cells revealed that the long transcript exhibited approximately 25% faster decay (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to total transcripts across all time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Similar stability patterns were observed in CAD cells expressing SNCA constructs with 3'UTR\u003csub\u003e290\u003c/sub\u003e or 3'UTR\u003csub\u003e2529\u003c/sub\u003e (data not shown).\u003c/p\u003e \u003cp\u003eThe impact of 3'UTR length on translation was evaluated through three complementary approaches. First, CAGG-driven SNCA constructs were expressed in Neuro2a cells, chosen for their absence of endogenous SNCA expression. Western blot analysis demonstrated progressively reduced protein production with increasing 3'UTR length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Second, fluorescence microscopy of CAD cells co-expressing EGFP CDS-SNCA 3'UTR hybrids containing the four main SNCA 3'UTR length variants with RFP as normalization control showed diminished EGFP fluorescence proportional to 3'UTR length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Third, dual-luciferase assays in SK-N-SH cells revealed that 3'UTR\u003csub\u003e2529\u003c/sub\u003e reduced luciferase activity by 30% compared to 3'UTR\u003csub\u003e575\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Consistent with reduced translation efficiency, RiboTag analysis in SK-N-SH cells expressing flagged RPL22 showed significantly decreased ribosomal association of long \u003cem\u003eSNCA\u003c/em\u003e transcripts compared to total \u003cem\u003eSNCA\u003c/em\u003e mRNA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Together, these approaches demonstrate that more extended SNCA 3'UTR variants exhibit reduced stability and translational efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDifferential subcellular localization of SNCA variants\u003c/h2\u003e \u003cp\u003eCombined RNA FISH and immunofluorescence analysis in differentiated CAD cells revealed distinct subcellular distribution patterns among SNCA variants. Longer transcripts showed increased cytoplasmic speckle formation, with 3'UTR\u003csub\u003e2529\u003c/sub\u003e displaying 70% more speckles than 3'UTR\u003csub\u003e290\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Moreover, longer variants (3'UTR\u003csub\u003e1075\u003c/sub\u003e or 3'UTR\u003csub\u003e2529\u003c/sub\u003e) exhibited preferential somatic localization, with 30% reduced axonal protein levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to shorter variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eThese findings demonstrate that SNCA 3'UTR length influences the spatial distribution of both transcripts and proteins, with longer variants showing distinct subcellular localization patterns that could impact local SNCA protein availability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ehnRNPA1 preferentially associates with SNCA 3'UTR and modulates alternative polyadenylation\u003c/h2\u003e \u003cp\u003eTo identify RBPs that interact with SNCA 3'UTR and regulate APA, biotinylated RNA spanning the 2,529 nt SNCA 3'UTR was synthesized and incubated with total brain lysates from postnatal day three mouse brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). RNP complexes were isolated using streptavidin-coated magnetic beads and analyzed by nano LC-MS/MS as an initial screening approach (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Differential analysis of proteins specifically enriched on SNCA 3'UTR beads compared to control beads was performed using GOTERM analysis in DAVID [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This revealed enrichment for 'nucleic acid binding' (27 proteins, 20.5%, Benjamini 4.2 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e), 'RNA binding' (29 proteins, 22.0%, Benjamini 5.6 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e), and 'double-stranded DNA binding' (12 proteins, 9.1%, Benjamini 3.4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The most enriched biological processes included 'mRNA processing' (17 proteins, 12.9%, Benjamini 3.2 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), 'RNA splicing' (14 proteins, 10.6%, Benjamini 2.8 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), and 'mRNA stabilization' (5 proteins, 3.8%, Benjamini 3.3 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). All RBPs detected in the SNCA 3'UTR\u003csub\u003e2529\u003c/sub\u003e pull-down analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, as even proteins showing some background binding could have physiologically relevant RNA interactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate their role in SNCA alternative polyadenylation regulation, 17 classical RBPs containing canonical RNA recognition motifs were selected from the identified RNA-binding proteins, cloned, and individually expressed in HEK293A cells. RT-qPCR analysis after 48 h revealed that hnRNPA1 most potently induced 3'UTR lengthening, doubling the ratio of long to total \u003cem\u003eSNCA\u003c/em\u003e transcripts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Analysis using ENCORI's platform showed strong anticorrelation between hnRNPA1 and \u003cem\u003eSNCA\u003c/em\u003e mRNA levels in lower-grade brain gliomas, suggesting a regulatory relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Given hnRNPA1's high expression levels, crucial role in RNA processing, and association with neurodegenerative diseases including familial ALS through its prion-like properties, it was selected for comprehensive analysis of its impact on SNCA expression.\u003c/p\u003e \u003cp\u003eThe interaction between hnRNPA1 and endogenous \u003cem\u003eSNCA\u003c/em\u003e mRNA was validated through RNA immunoprecipitation in SK-N-SH cells under native conditions (without UV crosslinking). Real-time RT-PCR analysis of RNA isolated from hnRNPA1 and control IgG immunoprecipitates showed six-fold enrichment of \u003cem\u003eSNCA\u003c/em\u003e mRNA in hnRNPA1 complexes, using \u003cem\u003eACTB\u003c/em\u003e mRNA, which contaminated all samples at low levels, as a normalization control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eTo map hnRNPA1 binding sites along \u003cem\u003eSNCA\u003c/em\u003e mRNA, four biotinylated RNA transcripts were generated spanning the 5'UTR, CDS, proximal (575 nt), and distal (2,000 nt) portions of the 3'UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, schematic). RNA-protein complexes were isolated from SK-N-SH lysates using these transcripts. Western blot analysis revealed that hnRNPA1 exclusively bound both proximal and distal portions of the SNCA 3'UTR, consistent with multiple predicted binding sites throughout these regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eCollectively, these findings establish hnRNPA1 as a key regulator of \u003cem\u003eSNCA\u003c/em\u003e mRNA processing through direct binding to multiple sites within the 3'UTR, with a particular impact on alternative polyadenylation and transcript variant distribution.\u003c/p\u003e \u003cp\u003e \u003cb\u003ehnRNPA1 enhances\u003c/b\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003etranscription\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven hnRNPA1's established role in transcriptional regulation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], its impact on \u003cem\u003eSNCA\u003c/em\u003e transcription was evaluated through gain- and loss-of-function experiments in SK-N-SH cells. Following 48 h of hnRNPA1 overexpression, RT-qPCR analysis of nuclear RNA revealed increased \u003cem\u003eSNCA\u003c/em\u003e pre-mRNA and mRNA levels across multiple regions: intron 3-exon 4 (61%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), intron 5-exon 6 (35%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and proximal exon 6 (3'UTR) (11%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). Conversely, simultaneous expression of two hnRNPA1 shRNA plasmids for 48 h resulted in only marginal decreases in \u003cem\u003eSNCA\u003c/em\u003e (pre-)mRNA levels, suggesting that reduced hnRNPA1 expression has a limited impact on \u003cem\u003eSNCA\u003c/em\u003e transcription.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results collectively establish hnRNPA1 as a positive regulator of \u003cem\u003eSNCA\u003c/em\u003e transcription, with elevated hnRNPA1 levels sufficient to enhance \u003cem\u003eSNCA\u003c/em\u003e mRNA expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003ehnRNPA1 modulates\u003c/b\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003etranscript distribution and local translation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe role of hnRNPA1 in \u003cem\u003eSNCA\u003c/em\u003e transcript localization was investigated, given its known nucleocytoplasmic shuttling capabilities. In SK-N-SH cells, modulating hnRNPA1 levels for 48 h did not affect the nuclear export of total \u003cem\u003eSNCA\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, hnRNPA1 overexpression specifically reduced the nuclear export of long \u003cem\u003eSNCA\u003c/em\u003e transcripts by 47% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while its depletion enhanced nuclear export by 38% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe impact of hnRNPA1 on SNCA ribonucleoprotein (RNP) complex formation and transport was further examined in differentiated CAD cells chosen for their extensive neuritic outgrowth. Co-expression of hnRNPA1 with SNCA constructs containing different 3'UTR lengths, followed by \u003cem\u003ein situ\u003c/em\u003e hybridization after 24 h, revealed distinct effects on RNP distribution. hnRNPA1 increased nuclear speckles for the shortest SNCA RNP complexes (370nt: 23%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and enhanced cytoplasmic speckle formation for shorter SNCA RNPs (370nt: 143%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 570nt: 232%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Additionally, hnRNPA1 significantly reduced axonal levels of longer \u003cem\u003eSNCA\u003c/em\u003e transcripts (570nt: -60%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 1075nt: -56%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 2550nt: -62%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), with corresponding decreases in axonal protein levels (570nt: -38%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 2550nt: -31%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThese findings demonstrate that hnRNPA1 differentially regulates \u003cem\u003eSNCA\u003c/em\u003e transcript distribution based on 3'UTR length, particularly affecting longer transcripts through reduced nuclear export, altered RNP complex formation, and decreased axonal localization and translation.\u003c/p\u003e \u003cp\u003e \u003cb\u003ehnRNPA1 regulates\u003c/b\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003emRNA stability and alternative polyadenylation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe role of hnRNPA1 in steady-state \u003cem\u003eSNCA\u003c/em\u003e mRNA levels was examined. Plasmid-mediated overexpression of hnRNPA1 in SK-N-SH and HEK293A cells for 48 h reduced total levels of endogenous \u003cem\u003eSNCA\u003c/em\u003e mRNA by approximately 30% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in both cell lines. Conversely, the expression of the long transcript increased by approximately 40% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in both cell lines. The ratio of the long over total SNCA transcripts was approximately 1.6 for both cell lines (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 for SK-N-SH and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for HEK293), indicating that excess hnRNPA1 induces 3'UTR lengthening and destabilizes \u003cem\u003eSNCA\u003c/em\u003e transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d). Notably, silencing hnRNPA1 expression by overexpressing hnRNPA1 shRNA plasmids for 48 h also decreased total \u003cem\u003eSNCA\u003c/em\u003e transcript levels by 48% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in SK-N-SH and 37% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in HEK293 cells. Lower hnRNPA1 expression significantly reduced the long \u003cem\u003eSNCA\u003c/em\u003e transcript levels in HEK293 (35%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but did not reach significance in SK-N-SH (25%, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) cells. Following hnRNPA1 downregulation, the long-over-total \u003cem\u003eSNCA\u003c/em\u003e transcripts ratio did not change in either cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-h).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand the mechanism behind these changes in steady-state levels, the stability of \u003cem\u003eSNCA\u003c/em\u003e transcripts was analyzed. Forty-eight hours after transfecting SK-N-SH cells with plasmids overexpressing or silencing hnRNPA1, actinomycin D treatment revealed that hnRNPA1 overexpression significantly decreased the half-life of total \u003cem\u003eSNCA\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In contrast, silencing hnRNPA1 increased total \u003cem\u003eSNCA\u003c/em\u003e mRNA expression at most time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Importantly, hnRNPA1 levels did not significantly affect the stability of the \u003cem\u003eSNCA\u003c/em\u003e transcripts with the long 3'UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). These experiments demonstrate that hnRNPA1 destabilizes \u003cem\u003eSNCA\u003c/em\u003e transcripts with shorter 3'UTRs, significantly downregulating total \u003cem\u003eSNCA\u003c/em\u003e mRNA levels. The regulatory effect is diminished on the longer \u003cem\u003eSNCA\u003c/em\u003e transcripts, possibly due to their increased nuclear localization following hnRNPA1 overexpression and interference from additional RBPs bound to the distal segment of SNCA's 3'UTR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mechanism by which hnRNPA1 induces \u003cem\u003eSNCA\u003c/em\u003e mRNA degradation was then investigated. Since previous studies have shown that hnRNPA1 can enhance the transcription or maturation of specific miRNAs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and miR-7 and miR-153 are known to regulate \u003cem\u003eSNCA\u003c/em\u003e mRNA expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the possibility that hnRNPA1 either enhances these miRNAs or promotes their recruitment on SNCA 3'UTR was examined. RT-qPCR analysis of miR-7 and miR-153 expression was performed on RNA extracted from SK-N-SH cells transfected with the plasmids overexpressing or silencing hnRNPA1. Mature miR-7 levels were increased by approximately 37% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while miR-153 expression was unaffected by hnRNPA1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). Next, a plasmid co-expressing miR-7 and miR-153 was co-transfected with the plasmids overexpressing or silencing hnRNPA1 in SK-N-SH cells. Forty-eight hours later, RT-qPCR analysis of \u003cem\u003eSNCA\u003c/em\u003e mRNA expression revealed that hnRNPA1 overexpression or silencing did not alter total or long \u003cem\u003eSNCA\u003c/em\u003e transcripts degradation mediated by miR-7/153 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h), suggesting that hnRNPA1 does not regulate the recruitment of miRISC-7/153 complexes on \u003cem\u003eSNCA\u003c/em\u003e mRNA and that it is dispensable for their function.\u003c/p\u003e \u003cp\u003eThe CCR4\u0026ndash;NOT (carbon catabolite repression 4-negative on TATA-less) complex, the major deadenylase in mammals, is formed by CNOT1 that acts as a scaffold to about seven subunits of which CNOT6 and CNOT7/CAF1 are the catalytic members. To investigate if hnRNPA1 directly recruits this complex on SNCA 3\u0026prime;UTR, the biotinylated SNCA 3'UTR\u003csub\u003e2529\u003c/sub\u003e was incubated with total lysates from SK-N-SH cells expressing varying quantities of hnRNPA1. Western blot analysis showed that hnRNPA1 did not pull CNOT1 or CNOT7 on SNCA 3\u0026prime;UTR (Supplementary Fig.\u0026nbsp;1). As the assay does not favor third-order interactions, the possibility that hnRNPA1, via interaction with another RBP, recruits CCR4\u0026ndash;NOT to \u003cem\u003eSNCA\u003c/em\u003e mRNA cannot be excluded [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePABPC1 binds to mRNAs' poly(A) tails, protecting them from shortening. To test if hnRNPA1 affected SNCA levels by altering PABPC1 levels, hnRNPA1 expression was modulated in SK-N-SH cells. Immunoblotting revealed that hnRNPA1 overexpression significantly decreased PABPC1 levels (31%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while lowering hnRNPA1 levels did not have an effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eThese data demonstrate that hnRNPA1 regulates \u003cem\u003eSNCA\u003c/em\u003e mRNA levels through multiple coordinated mechanisms. While promoting the decay of shorter transcripts in the cytoplasm through increased miR-7 expression and reduced PABPC1-mediated protection, the enhanced nuclear retention of longer transcripts shields them from these cytoplasmic degradation mechanisms. The observation that silencing hnRNPA1 leads to decreased steady-state \u003cem\u003eSNCA\u003c/em\u003e mRNA levels despite increased transcript stability suggests adaptation to prolonged hnRNPA1 depletion, where cells establish a new equilibrium with fewer but more stable transcripts.\u003c/p\u003e \u003cp\u003eSubsequently, the mechanism by which hnRNPA1 drives \u003cem\u003eSNCA\u003c/em\u003e mRNA alternative polyadenylation was investigated. In eukaryotes, cleavage and polyadenylation require specific interactions between cis-acting sequences and their corresponding protein complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The core polyadenylation signal (PAS), typically AAUAAA or AUUAAA, is recognized by CPSF30/CPSF4 and WDR33 of the polyadenylation specificity factor (CPSF) complex. Downstream of the PAS, CSTF64/CSTF2 of the cleavage-stimulating factor (CSTF) complex binds to a GU-rich downstream element (DSE). The interaction between CPSF and CSTF complexes is crucial for determining cleavage site selection. Additionally, the cleavage factor I (CFIm) complex, comprising NUDT21/CPSF5 and CFIm68/CPSF6, binds to U(G/A)UA motifs upstream of the PAS to facilitate cleavage site recognition. A fourth regulatory element, rich in G nucleotides and located downstream of the DSE, may also participate in polyadenylation through yet unidentified factors. To investigate whether hnRNPA1 influences APA by interfering with these core processing complexes, biotinylated SNCA 3'UTR\u003csub\u003e2529\u003c/sub\u003e was incubated with total lysates from SK-N-SH cells expressing increasing amounts of hnRNPA1. While the association of hnRNPA1 with SNCA 3'UTR increased as expected, the binding of CPSF, CSTF, and CFIm complexes remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), suggesting that hnRNPA1 does not directly compete with core APA machinery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnother potential mechanism involves the spliceosomal component U1 snRNP. Previous studies have shown that hnRNPA1 can directly interact with U1 snRNP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and reducing U1 snRNA levels can activate proximal polyadenylation sites [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, U1 snRNP complexed with RNA polymerase II can influence co-transcriptional recruitment of 3\u0026prime; processing factors, similar to mechanisms proposed for other RNA-binding proteins [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. RT-qPCR analysis following U1 snRNA depletion with two different siRNAs showed that despite 80% reduction in U1 snRNA levels, the ratio of long-to-total \u003cem\u003eSNCA\u003c/em\u003e transcripts showed a non-significant 10% increase, indicating that U1 snRNP is not essential for regulating SNCA APA site selection (data not shown).\u003c/p\u003e \u003cp\u003eThe role of transcription elongation rate in APA site selection was also examined, as increased pausing of RNA polymerase II (Pol II) downstream of an early PAS can enhance cleavage and polyadenylation at that specific site [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This mechanism is particularly relevant since proximal sites are encountered first by the APA machinery. hnRNPA1 is known to enhance transcription elongation by sequestering 7SK RNA, a repressor of the transcription elongation factor P-TEFb [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and its depletion leads to promoter-proximal pausing of RNA polymerase II on P-TEFb-dependent genes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Treatment of hnRNPA1-expressing SK-N-SH cells with DRB for 2 h, which blocks P-TEFb kinase binding to Pol II, followed by DRB removal, revealed that control cells showed the expected progressive increase in \u003cem\u003eSNCA\u003c/em\u003e transcripts. In contrast, hnRNPA1-transfected cells showed minimal response to DRB (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). This resistance to DRB treatment suggests maintained P-TEFb engagement with Pol II in the presence of hnRNPA1, supporting a model where hnRNPA1 promotes transcription elongation and consequently favors distal polyadenylation site usage in SNCA \u003cem\u003emRNA\u003c/em\u003e processing.\u003c/p\u003e \u003cp\u003eThese findings demonstrate that hnRNPA1 promotes distal polyadenylation site usage primarily through enhanced transcription elongation rather than direct interference with the core APA machinery or U1 snRNP-dependent mechanisms.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ehnRNPA1 regulates SNCA protein expression through multiple pathways\u003c/h2\u003e \u003cp\u003eThe impact of hnRNPA1 on SNCA protein expression was evaluated in SK-N-SH and HEK293A cells. Western blot analysis revealed that hnRNPA1 overexpression decreased total SNCA protein levels by 34% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) in SK-N-SH and 57% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in HEK293A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). To determine whether this decrease resulted from reduced mRNA levels or translation inhibition, ribosomal association of \u003cem\u003eSNCA\u003c/em\u003e mRNA was assessed using the RPL22-RiboTag assay in SK-N-SH cells. RNA-IP analysis showed that hnRNPA1 overexpression did not alter the association of total \u003cem\u003eSNCA\u003c/em\u003e transcripts with ribosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb), suggesting that excess hnRNPA1 reduces SNCA protein primarily through transcript destabilization. Unexpectedly, hnRNPA1 silencing increased total SNCA protein levels 2-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec) despite reducing steady-state \u003cem\u003eSNCA\u003c/em\u003e mRNA levels. RPL22-RiboTag analysis revealed a 50% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in ribosomal association of long \u003cem\u003eSNCA\u003c/em\u003e transcripts following hnRNPA1 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed), indicating enhanced translation efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSNCA protein is degraded through two major pathways: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Within the ALP, both macroautophagy and chaperone-mediated autophagy (CMA) are implicated in SNCA degradation, with LAMP2A as the SNCA receptor in CMA [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To explore whether hnRNPA1 affects SNCA protein levels by interfering with these catabolic pathways, its role in both systems was investigated.\u003c/p\u003e \u003cp\u003eThe UPS activity was assessed using the fluorogenic substrate Suc-LLVY-AMC, which releases fluorescent AMC upon proteasomal cleavage. hnRNPA1 overexpression enhanced proteasomal activity by 25% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while its silencing had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). These findings were validated using a GFP-based reporter system that allows quantification of ubiquitin-proteasome-dependent proteolysis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This system utilizes three constructs: Ub-R-GFP and Ub-GR76V-GFP, which incorporate N-end rule and ubiquitin fusion degradation signals, respectively, making them proteasome targets, and Ub-M-GFP, which upon ubiquitin cleavage remains stable like unmodified GFP. Western blot analysis showed that hnRNPA1 overexpression decreased R-GFP and GR76V-GFP levels by 50% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 27% (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), respectively, while its depletion increased these proteins by 60% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 98% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef), confirming that hnRNPA1 promotes UPS activity.\u003c/p\u003e \u003cp\u003eTo assess ALP function, p62 and LAMP2 levels were monitored, and while hnRNPA1 modulation did not affect their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg), autophagic flux was analyzed using a specialized reporter system. This system employs RlucLC3wt, which becomes degraded inside autophagosomes, and RlucLC3G120A, which cannot undergo lipidation and remains stable, serving as a control [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The analysis revealed that hnRNPA1 overexpression enhanced macroautophagy, while its depletion had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that hnRNPA1 reduces SNCA protein levels through multiple mechanisms: promoting transcript destabilization and degradation via enhanced UPS and macroautophagy activities, while its depletion increases SNCA protein by enhancing the ribosomal association of long transcripts and reducing UPS activity.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDysregulation of SNCA expression is a hallmark of several neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Understanding the mechanisms regulating SNCA expression is essential for therapeutic development. Previous research has established that the SNCA 5'UTR contains an internal ribosome entry site (IRES) element enabling protein synthesis during stress when most mRNAs are sequestered from translation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, SNPs in the SNCA 3'UTR associated with PD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and altered SNCA APA patterns in PD patients [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] highlight the significance of SNCA regulatory elements in disease pathogenesis.\u003c/p\u003e \u003cp\u003eThe current study demonstrates that \u003cem\u003eSNCA\u003c/em\u003e transcripts with longer 3'UTRs display distinct nuclear retention patterns, forming nuclear speckles and showing preferential somatic localization. The increased nuclear retention and accelerated decay of longer \u003cem\u003eSNCA\u003c/em\u003e transcripts, together with their reduced translation efficiency, indicate that alternative polyadenylation (APA) is an effective mechanism for modulating SNCA protein levels. This regulatory layer may be particularly relevant in neurons, where precise control of local protein synthesis is essential for synaptic function and plasticity.\u003c/p\u003e \u003cp\u003eIdentifying hnRNPA1 as a regulator of SNCA expression revealed multiple mechanistic control layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). hnRNPA1 binds directly to both proximal and distal segments of the SNCA 3'UTR and influences gene expression through distinct pathways. At the transcriptional level, hnRNPA1 enhances \u003cem\u003eSNCA\u003c/em\u003e mRNA expression, consistent with its previously described role in transcriptional regulation of other genes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The promotion of 3'UTR lengthening by hnRNPA1 appears to occur primarily through enhanced transcription elongation via P-TEFb rather than through direct interference with the core APA machinery or U1 snRNP-dependent mechanisms. This finding aligns with previous studies showing that transcription elongation rates can influence APA site selection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], although other processes, including epigenetic regulation through chromatin and histone modifications, cannot be excluded [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ehnRNPA1's impact on SNCA extends beyond transcription and APA (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The protein reduces the nuclear export of longer \u003cem\u003eSNCA\u003c/em\u003e transcripts while promoting the decay of shorter variants in the cytoplasm. This differential regulation coincides with increased miR-7 expression and reduced PABPC1 levels, suggesting that compartment-specific effects contribute to transcript-specific stability. The predominant role of cytoplasmic rather than nuclear degradation is supported by preliminary observations that depletion of nuclear exosome components DIS3 and DIS3L does not affect \u003cem\u003eSNCA\u003c/em\u003e transcript levels after hnRNPA1 overexpression (data not shown). During prolonged hnRNPA1 depletion, cells appear to adapt their RNA processing and decay machinery, reaching a new steady state with lower overall \u003cem\u003eSNCA\u003c/em\u003e mRNA levels but enhanced protection of remaining transcripts. Such temporal dynamics may be particularly relevant in conditions where chronic alterations in RBP levels occur.\u003c/p\u003e \u003cp\u003eAt the protein level, hnRNPA1 reduces SNCA abundance through multiple mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Beyond its effects on transcript levels and localization, hnRNPA1 enhances proteasomal activity and macroautophagy. This finding is particularly intriguing given that both the ubiquitin-proteasome system and autophagy-lysosome pathway are implicated in synucleinopathies [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. When hnRNPA1 is depleted, increased SNCA protein levels result from enhanced ribosomal association of long transcripts and reduced proteasomal function, as demonstrated by the RiboTag and proteolysis assays.\u003c/p\u003e \u003cp\u003eThe relationship between hnRNPA1 and neurodegenerative disease extends beyond its regulation of SNCA. Reduced hnRNPA1 levels have been reported in Alzheimer's disease and ALS cases with TDP43 aggregates [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and knockout hnRNPA1 mice exhibit cognitive dysfunction [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, overexpression of hnRNPA1 has been shown to reduce the formation of TDP43 aggregates [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Additionally, hnRNPA1's role in telomere biogenesis and maintenance [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] connects it to cellular aging mechanisms [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], a major risk factor for neurodegenerative disorders. In the context of this work, the hematopoietic- and neurologic-expressed sequence 1 (Hn1/JPT1) protein, which mitigates senescence phenotypes, is regulated by hnRNPA1 at the APA level [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In contrast to \u003cem\u003eSNCA\u003c/em\u003e mRNA, hnRNPA1 downregulation leads to 3'UTR lengthening of \u003cem\u003eHN1\u003c/em\u003e mRNA, resulting in decreased protein production [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], highlighting the context-dependent nature of hnRNPA1's regulatory effects.\u003c/p\u003e \u003cp\u003eThese findings open several important avenues for future research. The functional consequences of SNCA 3'UTR isoform expression need to be examined \u003cem\u003ein vivo\u003c/em\u003e, particularly in synaptic function and neurodegeneration. Additionally, determining whether hnRNPA1 expression is dysregulated in PD could provide new insights into disease mechanisms, especially given its role in regulating genes involved in cellular senescence and its connection to PD-associated SNPs in the SNCA 3'UTR.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study reveals that the SNCA 3'UTR governs critical regulatory events controlling alpha-synuclein expression and localization. Moreover, hnRNPA1 emerges as a central orchestrator of SNCA expression at both transcriptional and post-transcriptional levels, including APA, nuclear export, mRNA stability, and protein clearance. These insights advance our understanding of SNCA regulation and suggest that modulating hnRNPA1 or SNCA 3'UTR dynamics may provide therapeutic avenues for synucleinopathies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALP: autophagy-lysosome pathway; APA: alternative polyadenylation; CMA: chaperone-mediated autophagy; DLB: dementia with Lewy bodies; LB: Lewy bodies; hnRNPA1: A/U-rich element binding factor 1; IRES: internal ribosome entry site; MSA: multiple system atrophy; LC-MS/MS: liquid chromatography (LC) with mass spectrometry (MS); ns: not significant; PD: Parkinson\u0026apos;s disease; RBP: RNA binding protein; SNCA: alpha-synuclein; SNP: Single-nucleotide polymorphism; UPS: ubiquitin-proteasome system; UTR: untranslated region.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. G. Stoecklin (Center for Molecular Biology of Heidelberg University and Mannheim Institute for Innate Immunoscience of Heidelberg University, Germany) is acknowledged for providing the CNOT1 plasmid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Michael J. Fox Foundation for Parkinson\u0026apos;s Research (Grant ID16186), the Greek General Secretariat for Research and Innovation (\u0026Tau;2EDK-01291, TAEDR-0535850), and Empirikion Foundation (2021) to E.D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.D. designed primers and prepared plasmids. F.K. and E.D. conducted molecular and biochemical experiments. A.A.K. and G.T.T. performed proteomics experiments. F.K. and E.D. analyzed data. F.K. created graphs and diagrams. E.D. designed and supervised experiments and wrote the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article and the supplementary information files have included all generated data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [50] partner repository with the dataset identifier PXD060368.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRodent brain tissue was obtained in accordance with the European Union (2003/65/CE) guidelines regarding the use of laboratory animals. The experimental protocol was approved by both the Institutional Animal Care and Use Committee of BRFAA and the Veterinary Services of Attica prefecture (K/2134).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMa J, Gao J, Wang J, Xie A (2019) Prion-Like Mechanisms in Parkinson's Disease. 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Nucleic Acids Res 53:D543\u0026ndash;D553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkae1011\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkae1011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SNCA, hnRNPA1, RNA binding proteins, alternative polyadenylation, 3'UTR, mRNA stability","lastPublishedDoi":"10.21203/rs.3.rs-6294566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6294566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAbnormal accumulation of alpha-synuclein (SNCA) is a hallmark of Parkinson\u0026rsquo;s disease (PD), yet the molecular mechanisms governing SNCA expression remain incompletely understood. The SNCA 3' untranslated region (3'UTR) plays a critical regulatory role, with linkage disequilibrium suggesting its involvement in sporadic PD. Analysis of SNCA 3'UTR isoforms revealed that longer variants exhibit increased nuclear retention, form nuclear speckles, decay more rapidly, produce less protein, and preferentially localize to the soma rather than axons. Mass spectrometry identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a key regulator of alternative polyadenylation (APA), promoting longer SNCA 3'UTR isoforms. hnRNPA1 selectively binds proximal and distal regions of the SNCA 3'UTR but not the 5'UTR or coding sequence. Functional studies demonstrated that hnRNPA1 enhances SNCA transcription while reducing nucleocytoplasmic shuttling of longer 3'UTR isoforms, destabilizes shorter transcripts, and restricts axonal transport of longer variants. At the protein level, hnRNPA1 decreases SNCA expression by increasing proteasomal degradation and autophagic flux. These findings establish hnRNPA1 as a multifaceted regulator of SNCA, integrating transcriptional and post-transcriptional control via APA, mRNA stability, subcellular transport, and protein turnover.\u003c/p\u003e","manuscriptTitle":"hnRNPA1 orchestrates multi-layered regulation of SNCA expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 16:52:23","doi":"10.21203/rs.3.rs-6294566/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b55d3ab2-8756-4632-9653-c99a2f2afa23","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T08:42:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-03 16:52:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6294566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6294566","identity":"rs-6294566","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}