Extension of mRNA poly(A) tails and 3′UTRs during neuronal differentiation exhibits variable association with post-transcriptional dynamics

Abstract Differentiation of neural progenitor cells into mature neuronal phenotypes relies on extensive temporospatial coordination of mRNA expression to support the development of functional brain circuitry. Cleavage and polyadenylation of mRNA has tremendous regulatory capacity through the alteration of mRNA stability and modulation of microRNA (miRNA) function, however the extent of utilization in neuronal development is currently unclear. Here, we employed poly(A) tail sequencing, mRNA sequencing, ribosome profiling and small RNA sequencing to explore the functional relationship between mRNA abundance, translation, poly(A) tail length, alternative polyadenylation (APA) and miRNA expression in an in vitro model of neuronal differentiation. Differential analysis revealed a strong bias towards poly(A) tail and 3′UTR lengthening during differentiation, both of which were positively correlated with changes in mRNA abundance, but not translation. Globally, changes in miRNA expression were predominantly associated with mRNA abundance and translation, however several miRNA–mRNA pairings with potential to regulate poly(A) tail length were identified. Furthermore, 3′UTR lengthening was observed to significantly increase the inclusion of non-conserved miRNA binding sites, potentially enhancing the regulatory capacity of these molecules in mature neuronal cells. Together, our findings suggest poly(A) tail length and APA function as part of a rich post-transcriptional regulatory matrix during neuronal differentiation.


INTRODUCTION
The generation of neuronal subpopulations with unique morphological and electr ophysiological pr operties is dri v en by discrete patterns of mRNA expression in response to developmental cues. It is currently well established that activation of neuron-specific mRNA expression is largely mediated by an array of transcription factors, that modify the transcriptome throughout the course of the differentiation process (1)(2)(3). In contrast, our knowledge of the posttranscriptional regulatory systems which fine-tune mRNA expression during neuronal dif ferentia tion is less comprehensi v e. Although a vast collection of cis -and trans -acting mechanisms contribute to mRNA localisation, stability and translational capacity during de v elopment and differentiation (4)(5)(6), the behaviour and interplay of these mechanisms remains elusi v e. This is significant, gi v en that dysregulation of post-transcriptional systems has emerged as a common feature amongst se v eral psy chiatric disor ders associated with disrupted neuronal de v elopment, such as schizophrenia, bipolar disorder and autism spectrum disorders ( 7 , 8 ).
One dimension of mRN A biolo gy thought to be pivotal in multiple aspects of post-transcriptional regulation is cleavage and polyadenylation, a near ubiquitous mRNA end processing mechanism intricately coupled with transcriptional termination. Canonicall y, pre-mRN A cleavage is initiated downstream of conserved polyadenylation site motifs, of which the A[A / U]UAAA motif is most prominent, following which poly(A) polymerase synthesises a poly(A) tail of approximately 50-100nt ( 9 , 10 ). An intact poly(A) tail is critical throughout the life cycle of an mRNA, gi v en this structure is r equir ed for mRNA nuclear export ( 11 ), confers resistance to degradation ( 12 ) and enables association with poly(A) binding proteins for enhancement of translational efficiency ( 13 ). Furthermore, erosion of the poly(A) tail by CCR4-NOT and P AN2-P AN3 complexes constitutes the predominant mechanism through w hich microRN As (miRN As) instiga te degrada tion of partially complementary mRNAs in eukaryotic cells (14)(15)(16). In neurons, cytoplasmic polyadenylation may also regula te transla tional competency of mRNAs in close proximity to the synapse ( 17 , 18 ). While these facets of poly(A) tail dynamics suggest a particularly important role in posttranscriptional regulation, global changes in polyadenylation and their association with mRNA dynamics and miRNA expression during neuronal dif ferentia tion are yet to be characterised.
Alternati v e polyadenylation (APA) can also drastically impact post-transcriptional dynamics by modifying utilisa tion of polyadenyla tion signals (PAS) (19)(20)(21)(22)(23). These changes directly affect 3´UTR length and subsequently impact key regulatory motifs such as AU-rich elements ( 24 ), localisation elements ( 25 ) and miRNA binding sites ( 26 ), all of which contribute to mRNA stability and translational status ( 27 ). Interestingly, cell-specific APA profiles have been recently discovered, with proliferative cells generally expressing short 3´UTR APA isoforms ( 28 ), whereas complex, terminally dif ferentia ted cells such as neurons are often associated with longer 3´UTRs ( 29 ). During neuronal dif ferentia tion, the functional consequences of APA have recently been described for specific genes such as Bdnf ( 30 ) and Rac1 ( 31 ), while emerging studies have identified a transition towards long 3´UTR expression during neuronal dif ferentia tion ( 32 , 33 ) and brain de v elopment ( 34 , 35 ), indicating a particularly important role in this phenotypic transition.
The recent de v elopment of high-throughput strategies for poly(A) tail and 3´UTR quantification ( 36 , 37 ) now provide an opportunity to explore these broad regulatory mechanisms on a genome-wide scale. To this end, we employed genome-wide poly(A) tail sequencing (PAT-Seq) ( 36 ), mRN A sequencing (mRN A-Seq), ribosome profiling (Ribo-Seq) and small RNA sequencing to gain further insight into the regulation of mRNA cleavage and polyadenylation during neuronal differentiation, and their interplay with mRNA and miRNA dynamics. Our study revealed many genes subjected to poly(A) tail and / or 3´UTR lengthening during neuronal dif ferentia tion, both of which were associated with mRNA expression le v els. We also identify miRN A-mRN A pairings associated with pol y(A) tail length modulation and explore the impact of 3´UTR lengthening on miRNA binding sites. These results suggest lengthening of poly(A) tails and 3´UTRs constitutes an important mechanism of post-transcriptional mRNA regulation associated with neuronal differentiation.

Neuronal differentiation
Neuronal dif ferentia tion was induced via sequential trea tment with all-trans retinoic acid (ATRA, Sigma-Aldrich, St. Louis, MO, USA) and brain-deri v ed neur otr ophic factor (BDNF, Life Technologies, Carlsbad, CA, USA), as previously described ( 38 ). One day prior to dif ferentia tion (day -1), cells were seeded into T75 culture flasks at a density of 25 000 cells / cm 2 in standard culture medium. The next day (day 0), standard medium was replaced with ATRA-supplemented medium (10 M) and cells were pr otected fr om light exposur e, with ATRA medium r eplaced on day 2. On day 5, ATRA medium was aspirated, cells were washed 3 times with standard culture medium, then returned to standard culturing conditions in BDNF-supplemented (50ng / mL), serum-free medium. BDNF medium was replaced on days 7, 9 and 11, after which the methods were continued as described on day 12. Successful neuronal dif ferentia tion was established by examining neurite outgrowth during ATRA / BDNF treatment and analysing the expression of neuronal marker genes (see Figure 1 B-E). Dif ferentia ted cells were compar ed to untr eated control samples cultur ed under standard conditions.

PA T -seq library preparation
Total RNA was extracted from all samples by harvesting cells in 1mL TRIzol reagent (ThermoFisher, Waltham, MA, USA), as per the manufacturer's instructions. Poly(A) tail sequencing (PAT-Seq) was conducted using 1 g of high-quality RN A (RN A integrity number = 10) as previously described ( 36 ). Briefly, biotinylated ePAT primers (5´-Bio-CAGACGT GT GCT CTT CCGAT CT (18) -3´; 100 M) were annealed to polyadenylated mRNAs and 3´ends were extended using 5 units (U) of the Klenow fragment of Figure 1. Sequencing of neuronally dif ferentia ted SH-SY5Y cells. ( A ) Schema tic ov ervie w of the neuronal dif ferentia tion par adigm and sequencing str ategies employed in this study. (B-D) Phase contr ast microgr aphs depicting SH-SY5Y cells cultured under standard conditions ( B ), 5 days after media supplementation with ATRA ( C ), and after ATRA treatment followed by an additional 7 days of serum starvation and BDNF treatment ( D ). Note the e xtensi v e de v elopment of neurites after dif ferentia tion rela ti v e to na ïve cells (white arrowheads). Scale bar = 100 m. ( E ) Expression of known neuronal marker genes in control (purple) and dif ferentia ted (green) cells, as quantified via mRNA sequencing. All genes were subjected to statistically significant upregulation (Benjamini-Hochberg FDR < 0.05), with CACNA1C , FOS , NTRK2 and RELN additionally subjected to > 2-fold incr eased expr ession r elati v e to na ïv e cells. Data presented as log 2 counts-per-million (CPM) ± SD. ** FDR < 0.01, **** FDR < 0.0001. ( F ) Genomic distribution of uniquely aligned reads for mRNA-Seq, PAT-Seq and Ribo-Seq libraries. Data presented as assigned tags per kilobase per million assigned tags ± SD. ( G ) Metagene analysis of read density around CDS start and stop codons. Note that PAT-Seq libraries exhibit incr eased r ead density in a 5´to 3´direction, particularly around CDS stop codons. ( H ) Sub-codon phasing of all three libraries. Note that Ribo-Seq reads exhibit bias for the first reading frame as expected for ribosome-protected fragments. ( I ) Correlation of gene-le v el read-count profiles amongst biological replicate samples. One replicate from the dif ferentia ted group was excluded from the Ribo-Seq experiment due to poor data quality. DN A pol ymerase I (NEB, Ipswich, MA, USA). RNAs were then subjected to limited RNase T 1 digestion (10U, Roche, Basel, Switzerland), with parameters optimised to produce ∼250nt fragments, after which 3´fragments were collected with streptavidin beads (NEB). Phosphorylation of 5´-ends was next performed with poly nucleotide kinase (0.5U, NEB), prior to ligation of 5´splinted linkers, re v erse transcription, and size selection (120-400 bp) via 6% denaturing urea-polyacrylamide gel. To amplify all samples, 10 l of cDNA was subjected to 16 PCR cycles, with 2 l of ScriptSeq index ed r everse primers (

Quantification of poly(A) tail length and APA events
PAT-Seq data were analysed using the tail tools (v.0.41) R package ( 36 ). Firstl y, ada pter sequences and poly(A) tails were trimmed from fastq-formatted reads. Poly(A) tails were identified by calling a run of consecuti v e 'A' residues extending to the 3´end, or the adapter sequence, with an error rate of 1 in 5 bases and Phred33 quality score > 10 allow ed. Reads w er e then aligned to the r efer ence genome (hg19, UCSC) using Bowtie2 ( 39 ) and deemed polyadenylated if a non-templated run of ≥ 4 consecuti v e 'A' residues was detected. For genes with ≥ 10 poly(A) reads, average tail length was calcula ted. Dif ferential analysis of tail length between experimental conditions was determined using the limma ( 40 ) voom statistical pipeline, accounting for depthassocia ted varia tion in tail length estima tions. Benjamini-Hochberg false discovery rate ( FDR BH ) < 0.1 and absolute change in tail length > 10 'A' r esidues wer e consider ed significant.
APA e v ents were identified with the DEXSeq (v1.38.0) R package ( 41 ). Poly(A) reads overlapping known MACEsupported 2-30nt APA sites curated from APADB (v2) ( 21 ) were counted using the dexseq count.py script. Counts were then converted into a DEXSeq dataset, after which library size factors, dispersions and APA fold changes were estimated. Sites with ≥2 normalised counts across all samples wer e r etained, after which differ ential APA site usage between control and dif ferentia ted cells was determined via likelihood ra tio test. Rela ti v e e xpr ession differ ence (RED) between the two most abundant APA sites (i.e. proximal and distal) across both conditions was then determined as follows: Significant lengthening e v ents r equir ed RED > 1, coupled with significantly increased ( FDR BH < 0.1) expression of the distal isoform and / or a significant decrease of the proximal isof orm. Con v ersely, significant shortening e v ents r equir ed a RED < -1, with a significant decrease in expression of the distal isoform and / or a significant increase of the proximal isoform. APA e v ents were validated via qPCR utilising primers designed to amplify the entire gene, or the long 3´UTR isoform only (see Supplementary Methods for further details, and Supplementary Table S1 for primer sequences). mRNA sequencing, ribosome profiling and small RNA sequencing mRNA libr ary prepar ation was conducted using the Illumina TruSeq Str anded mRNA Libr ary Prepar ation Kit, according to the manufacturer's instructions. Briefly, 1 g of total RNA was subjected to poly(A) selection via oligo-DT beads, hea t fragmenta tion (94 • C , 4 min), re v erse transcription, adapter ligation and amplification via 15 PCR cycles. Libraries were subjected to 151 cycles of single end sequencing using the Illumina NextSeq500 platform.
Ribosome profiling (Ribo-Seq) was performed via the Illumina TruSeq Ribo Profile Kit (H / M / R), with minor amendments. Translational activity was firstly suspended by incubating cells in warm culture medium supplemented with 0.1mg / ml cy clohe ximide (CHX) for 1 min. Cells were harvested on ice with 800 l mammalian lysis buffer (containing 0.1mg / ml CHX and 25U Turbo DNase I (Ther-moFisher)), and ribosome foot-printing was conducted by adding 150U RNase I (Invitro gen) to 300 l clarified l ysate for 45 min. Library preparation was conducted per the manufacturer's instructions, with libraries amplified via 12 PCR cycles and subjected to 101 cycles of single end sequencing via a NovaSeq6000 Instrument.
For small RNA sequencing, libraries were produced from 1 g of total RNA using the Illumina TruSeq Small RNA Libr ary Prepar ation Kit, according to the manufacturer's instructions. Firstl y, ada pters were ligated to RNAs, prior to re v erse transcription and amplification via 11 PCR cycles. Samples were pooled in equimolar ratios and volumes, and small RNAs between ∼20-33nt were purified via 6% nati v e PAGE. The resultant library was subjected to 76 cycles of single end sequencing using an Illumina NextSeq500. See Supplementary Methods for further details.
For small RNA data, libraries were firstly aligned to human precursor and mature tRNAs curated from gtR-NAdb (v2.0) ( 48 ) using Bowtie2 , for identification of tRNAderi v ed small RNAs. Unaligned reads were mapped to the NCBI GRCh38 r efer ence genome using Bowtie2 . This r eference genome assembly was selected to ensure compatibility Nucleic Acids Research, 2023, Vol. 51, No. 15 8185 with the miRBase (release 22) ( 49 ) mature miRNA annotation file during read-counting via HTSeq . See Supplementary Methods for further details.

Differ ential expr ession analysis
Da ta normalisa tion and dif fer ential expr ession wer e conducted with edgeR (v3.8) ( 50 ). Raw counts for each sample were merged into a single matrix and normalised to sequencing depth (counts-per-million; CPM). Genes and miRNAs with consistently low counts were then removed by employing a minimum CPM threshold, ensuring genes / miRNAs with < 5 raw counts in the smallest library were excluded. Sample clustering and biological variation were assessed by visually inspecting multidimensional scaling (MDS) and biological coefficient of variation (BCV) plots (Supplementary Figures S1, S2). After calculating normalisation factors (TMM method) and estimating dispersion, differ ential expr ession between tr eated and control samples was determined via pairwise exact test, with FDR BH < 0.05 and absolute log 2 fold change (log 2 FC) > 1 consider ed significant. Differ ential translational efficiency was determined via RiboDiff (v.0.2.1) ( 51 ). See Supplementary Methods for further details.

Gene ontology analysis
Gene ontology enrichment was analysed with the Toppgene functional enrichment w e b suite ( 52 ), with FDR BH < 0.05 considered significant.

Sequencing of neuronally differentiated SH-SY5Y cells
To investigate 3´UTR dynamics associated with neuronal dif ferentia tion, poly(A)-tail sequencing (PAT-Seq), mRNA sequencing (mRNA-Seq) and ribosome profiling (Ribo-Seq) were conducted on SH-SY5Y cells sequentially treated with all-trans retinoic acid (ATRA) and brain-deri v ed neur otr ophic factor (BDNF), and untreated controls ( Figure  1 A). Successful neuronal dif ferentia tion was confirmed by examining neurite outgrowth, reduced proliferation, and the acquisition of a polar cellular morpholo gy, w hich was later supported by the observed upregulation of known neuronal marker genes via mRNA-Seq (Figure 1 B-E).
The PAT-Seq methodology produced an average of 6.07 × 10 6 ( ±2.83 × 10 6 ) polyadenylated ( ≥ 4nt) reads per sample, which pr efer entially aligned to 3´UTRs, consistent with a library preparation that constrains reads to the last ∼200 bases of the 3´UTR and poly(A) tail (Figure 1 (Figure 1 G). For Ribo-Seq libraries, sub-codon phasing additionally re v ealed an alignment pr efer ence for the first r eading frame, indicati v e of ribosomal triplet periodicity (Figure 1 H). Finally, comparison of gene-le v el read-counts re v ealed strong correlation ( R 2 ≥ 0.84) between biological replicates for all three library preparation methods, except for replicate 'dif3' in the Ribo-Seq analysis, which was excluded herein (Figure 1 I).

Poly(A) tail lengthening during neuronal differentiation
We next analysed changes in mRNA poly(A) tail length across 13021 genes with a detectable poly(A) tail. Across the tr anscriptome, aver age poly(A) tail length was significantly increased in dif ferentia ted cells (47.34 A residues) relati v e to controls (43.59 A residues), suggesting neuronal dif ferentia tion is associated with global poly(A) lengthening ( P < 0.0001, Kolmogoro v-Smirno v test;  Table S4).
To explore the functional significance of polyadenylation during neuronal dif ferentia tion, we examined cellular properties after 5 days of ATRA treatment in the presence of cor dy cepin (CDY), an adenosine deri vati v e known to pr ematur el y terminate pol yadenylation ( 55 , 56 ). A trunca ted dif ferentia tion paradigm was employed to capture changes during, rather than after, this phenotypic transition. Across three sub-IC 50 ( 57 ) Figure S3B). Collecti v ely, these results suggest that while poly(A) tail lengthening during neuronal dif ferentia tion predominantly af fects genes associa ted with this phenotypic transition, pharmacological inhibition of pol yadenylation a ppears to exert minimal impacts on neuronal dif ferentia tion.

Increased poly(A) tail length is associated with enhanced mRN A e xpression
We ne xt e xplored interplay between poly(A) tail length, mRNA expression and translation during neuronal  Table S11).
The behaviour of mRNAs targeted by differentially expressed miRNAs was explored using in silico miRNA-mRNA predictions from TargetScan v7.2 ( 59 ), exclud-ing 31 miRNAs deemed potentially misannotated by the TargetScan database, as well as low confidence mRNA-miRN A pairings with cum ulati v e weighted conte xt++ score > -0.2. Genes targeted by upregulated miRNAs exhibited decreased translation and transla tional ef ficiency, howe v er no consistent changes in mRNA expression nor poly(A) tail length survi v ed correction for multiple testing (Figure 4 C, D, top 5 most significant miRNA target gene profiles shown). Similarly, genes targeted by downregulated miRNAs displayed incr eased mRNA expr ession, translation and transla tional ef ficiency, but no significant alteration of poly(A) tail length (Figure 4 E, F). These findings were largely consistent after analysing the combined effect of all upregulated or downregulated miRNAs, stratified the number of predicted miRN A reco gnition elements for each gene (Supplementary Figure S5). We also investigated post-transcriptional dynamics associated with tRNAderi v ed small RNA fragments, a class of short non-coding RN A related to miRN As ( 60 , 61 ), howe v er no consistent changes were observed (Supplementary Figure S6).
To probe specific miRN A-pol y(A) tail length associations, negati v el y correlated miRN A-mRN A pairings were anal ysed with miR Comb ( 62 ). We identified 49 significant correlations between 24 differ entially expr essed miRNAs and 36 mRNAs ( FDR BH < 0.05; Figure Tables S13, S14). Interestingly, a subnetwork consisting of 22 genes and 11 miRNAs was uncovered, howe v er these genes exhibited no significant enrichment of GO annotations (Figure 5 D). Collecti v ely, these findings suggest that w hile miRN A expression is generall y associated with mRNA expression and translation during dif ferentia tion, any observable effect on poly(A) tail length may be confined to specific miRN A-mRN A pairings in our paradigm.

Increased usage of distal polyadenylation signals during neuronal differentiation
Recent studies suggest alternati v e polyadenylation is highly dynamic during long-term cellular processes such as differentiation ( 32-35 ) (Figure 6 A). To establish whether alternati v e polyadenylation (APA) was prevalent during neuronal dif ferentia tion, changes in polyadenyla tion signal (PAS) usage were determined from the PAT-Seq data using DEXSeq ( 41 ). Overall, 22898 PASs across 11213 genes were identified, with more than 45% of genes containing > 1 expressed PAS (Figure 6 B). A pproximatel y 87% PASs were identified within 3´UTRs as anticipated, whereas ∼7% were downstream of the most 3´exon ('extensions'), and a further ∼4% expressed fr om intr onic r egions (Figur e 6 C). Notably, the genomic distribution of expressed PASs significantly differed between both conditions ( P < 0.0001, Chi-squared test), indicating potential for differential regulation during neuronal dif ferentia tion (Figure 6 C).
Gene le v el changes in PAS usage were ne xt e xamined to identify significant APA e v ents. To simplify this analysis, the    top two most expressed PAS were analysed for each gene, noting that we predominantly focused on 3´UTR PASs, which possess the highest capacity to modulate miRNA binding sites and other regulatory elements. Differential analysis re v ealed 118 genes with significant APA e v ents, of which 110 (93%) were associated with 3´UTR lengthening (Figure 6 D, see Figure 6 E for r epr esentati v e lengthening e v ent, Supplementary Tab le S15). Fi v e genes were selected for qPCR validation using primer sets targeting the alternati v e 3´UTR and a common upstr eam r egion, of which two ( PNPLA4 , NANP ) were significantly lengthened ( FDR < 0.01) and two ( PSMA5 , ATP6AP2 ) trended towards lengthening ( FDR = 0.074, Figure 6 F). Quantification of 3´UTR length changes re v ealed lengthened genes were subjected to a median increase of 1,517nt, whereas shortened genes exhibited a median decrease of 903nt (Figure 6 G). In contrast, the remainder of the transcriptome increased by a median of 100nt (Figure 6 G). Surprisingly, GO term analysis re v ealed no consistent patterns of enrichment upon analysis of significantly lengthened and shortened genes, indicating genes affected by APA e v ents were functionally di v erse (Supplementary Tab le S16).

Effect of 3´UTR lengthening on mRN A d ynamics and miRNA binding sites
Since 3´UTR length changes are thought to impact critical regulatory motifs (24)(25)(26), the effect of APA e v ents on mRN A expression, translation, and pol y(A) tail length wer e explor ed. These analyses wer e r estricted to significant lengthening e v ents, as shortening e v ents impacted an insufficient number of genes for reliable statistical analysis. Overall, 3´UTR lengthening was significantly correlated with mRNA expression ( r = 0.36, P = 6.43 × 10 −5 ) and poly(A) tail length ( r = 0.21, P = 0.032), but not mRNA translation ( r = 0.03, P = 0.293) or translational efficiency ( r = -0.13, P = 0.087; Figure 6 H). Interestingly, genes with lengthened 3´UTRs were also subjected to increased poly(A) tail length, howe v er the magnitude of poly(A) lengthening was lower than genes unaffected by APA ( P = 0.037; Supplementary Figur e S7). Furthermor e, the top 50% of lengthened genes were biased towards increased mRNA expr ession, wher eas the lower 50% of genes exhibited decr eased expr ession ( P Bonferroni ≤ 0.0219 for all comparisons, Kruskal-Wallis test with Dunn's post-hoc multiple comparisons test; Supplementary Figure S7). Together, these results suggest that 3´UTR extensions via APA during neuronal dif ferentia tion are associated with mRNA expression and poly(A) tail length.
Howe v er, genes targeted by upregulated and downregulated miRNAs exhibited no significant bias towards lengthening or shortening compared to the transcriptome (Figure 7 D).

Differ ential expr ession and tr anslation of genes associated with polyadenylation
We ne xt e xamined the e xpression and translation of genes potentially associated with regulation of poly(A) tail and 3´UTR length during neuronal dif ferentia tion. Utilising GO annota tions associa ted with cleavage and polyadenylation and deadenylation (MSigDB ( 63 )), 29 genes were identified with significant changes at the mRNA and translational le v els (Figure 8 A). Notab ly, none of these 29 genes exhibited significant alteration of poly(A) tail length, nor APA. Interestingly, robust transcriptional and translational upregulation was identified for CELF2 , a known regulator of alternati v e polyadenylation e v ents ( 64 ). Dual upregulation was also observed for the Poly(A)-Binding Protein C5 ( PABPC5 ), brain-enriched Cytoplasmic Polyadenylation Element Binding Proteins CPEB3 and CPEB4 , and neuron-specific ELAV-like RNA Binding Proteins ELAVL2 and ELAVL4 . Since neuronal ELA VL (nELA VL) proteins have been previously associated with multiple aspects of mRNA metabolism ( 65 ), we examined the behaviour of nELAVL target genes utilising CLIP-Seq data from human brain samples ( 65 ). Interestingly, the number of nELAVL 3´UTR peaks exhibited a cumulative relationship with mRNA expression ( P Bonferroni ≤ 1.79e −6 , Kolmogoro v-Smirno v test), while genes with ≥1 nELAVL site were subjected to translational enhancement versus genes with no site ( P Bonferroni ≤ 8.8e −16 ; Figure 8 B). Furthermore, higher le v els of nELAVL peaks were associated with poly(A) tail ( P Bonferroni ≤ 8.25e −3 for genes with ≥ 6 peaks) and 3´UTR lengthening ( P Bonferroni ≤ 1.37e −5 for genes with ≥2 peaks, Figure 8 B).

DISCUSSION
The complex and dynamic network ar chitectur e in the brain is established and regulated though its neuronal nodes. These features are made possible by the discrete tempor ospatial contr ol of RNA translation that is mediated by a plethora of interacting post-transcriptional regulatory mechanisms. In the current study, we explored the interplay between se v eral of these processes during neuronal differentiation using a variety of RNA sequencing strategies, to characterize mRNA 3´UTR dynamics and their interrelationship with mRNA abundance, translation, and miRNA. Our findings suggest modulation of poly(A) tail length and APA function as part of a rich regulatory matrix, enabling d ynamic coordina tion of mRNA during dif ferentia tion.  Transcriptome-wide quantification of poly(A) tail length re v ealed a bias towards poly(A) lengthening during neuronal dif ferentia tion, w hich predominantl y affected genes enriched for dif ferentia tion-associa ted functions. Interestingly, many genes with increased poly(A) tail length also exhibited upregulation of steady-state mRNA le v els in a manner which disproportiona tely af fected genes annotated to the syna pse, w hereas translational profiles for the same genes remained largely unchanged. The expression of long poly(A) tails in eukaryotic cells is known to support association with poly(A) binding proteins, which establish 5´to 3´mRNA interactions important for mRNA stability and translational activity ( 12 , 13 , 66 ). While changes in polyadenylation and their relationship with mRNA expression have not been observed previously during neuronal dif ferentia tion, recent studies have shown that poly(A) tail lengthening in de v eloping oocytes enhances stability and translation of mRNAs associated with embryonic de v elopment ( 53 , 67 , 68 ). Strikingly, emerging evidence suggests poly(A) tail length and cytoplasmic poly(A) binding protein, PABPC , function as key mediators of translational enhancement in oocytes and early embryos, whereas mRNA stability is favoured in comparati v ely mature cell types, underscoring the importance of polyadenylation as a critical de v elopmental switch ( 69 ). These studies are consistent with the observed association between poly(A) tail lengthening and mRNA expression, but not translation, in the current study. It is plausible that poly(A) tail lengthening may exert a more-robust effect on neuronal translation in response to acute stimuli such as neuronal activation, as observ ed pre viously in paradigms of long-term potentiation ( 17 , 70 ). Nonetheless, we suspect poly(A) tail lengthening may contribute to neuronal dif ferentia tion by enhancing the abundance and stability of mRNAs important for this phenotypic transition, howe v er further e xploration is r equir ed to dissect the significance of poly(A) lengthening and increased mRNA expression in the absence of enhanced translational activity.
Anal ysis of miRN A expr ession r e v ealed substantial alteration of miRNA profiles during neuronal dif ferentia tion, including upregulation of neuronally enriched miRNAs, such as miR-132-5p and miR-212-5p, consistent with previous analysis of ATRA / BDNF dif ferentia ted SH-SY5Y cells ( 58 ). Interestingly, genes targeted by differentially expr essed miRNAs wer e subjected to alter ed mRNA expr ession, tr anslation and tr anslational efficiency in a manner directionally consistent with the canonical model of miRNA function, characterised by sequential deadenylation, decapping and exonucleolytic degradation of target mRNAs (14)(15)(16). Similar profiles of miRNA-associated mRNA r epr ession have been previously observed on a transcriptomewide scale after repea ted depolarisa tion of neuronally differentiated cells ( 71 ). Contrary to e xpectation, howe v er, associations between miRNA and poly(A) tail length wer e r estricted to a small subset of miRN A-mRN A correlations. We suspect transcriptome-wide miRNA-dependent regulation of the poly(A) tail may be more readily observed during early stages of neuronal dif ferentia tion, during which post-transcriptional remodelling of mRNA temporospatial dynamics is likely to be prevalent, thus necessitating future studies with greater temporal resolution.
A clear bias towards 3´UTR lengthening was additionally uncover ed, consistent with pr evious studies of neuronal differentiation and de v elopment (32)(33)(34)(35). Interestingly, the diverse functional r epr esentation of lengthened genes has also been observed in a subset of these studies ( 32 , 33 ), suggesting that discrete APA e v ents hav e functional significance at the individual gene level. We also identified positive correlation between 3´UTR lengthening, mRNA expression and poly(A) tail length, a surprising finding gi v en that 3´UTR lengthening is thought to enhance the inclusion of cis -acting regulatory elements associated with mRNA destabilization, particularly AU-rich elements and miRNA binding sites ( 23 , 24 , 26 ). Indeed, over 13000 miRNA binding sites were gained via 3´UTR lengthening in the current stud y. W hile this contradicts the canonical mechanism of miRNA function, it is plausible that many of these sites do not conform to conventional mechanisms of miRNA r epr ession, or ar e not well adapted, gi v en the vast majority of affected sites were non-conserved and affected non-conserved miRNA families ( 71 ). This consequently r epr esents an exciting avenue for further mechanistic exploration, in which key miR-NAs uncovered in the current study -such as miR-532-3p and miR-340-5p -may yield novel insights. We also cannot exclude the possibility that APA-associated changes in 3´UTR length may serve to control the subcellular localisa tion of af fected mRNAs in dif ferentia ted neurons, considering localisation elements ar e pr edominantly confined to the 3´UTR ( 72 ). Finally, we note that APA sites identified from the mRNA-Seq data using DaPars2 ( 28 ) exhibited weak correlation with APA e v ents identified from the PAT-Seq data (Pearson r = 0.056, P = 1.1 × 10 -4 , Supplementary Figure S8, Table S19). Furthermore, only one of fiv e DaPar s2 -nomina ted sites selected for qPCR was successfully validated, suggesting 3´-enriched sequencing methods may exhibit greater specificity for APA analysis than conventional mRNA-Seq (Supplementary Figure S8, Table S19).
Interestingly, se v eral genes involved in regulation of pre-mRNA cleavage, alternati v e polyadenylation and poly(A) tail binding were differentially expressed and translated in the current study, which may underpin the observed changes in 3´UTR regulation. Notably, we identified transcriptional and translation upregulation of the neuronspecific ELAVL2 and ELAVL4 genes, members of the ELAVL family of AU-rich element RNA binding proteins associated with neuronal differentiation and de v elopment ( 73 , 74 ), as well as mRN A stability, translation, pol yadenylation and splicing ( 65 , 75 ). Strikingl y, experimentall y supported nELAVL target genes exhibited significantly altered mRNA expression, translation, poly(A) tail length and 3´UTR length in the current study, consistent with previous evidence ( 65 , 75 ). We additionally identified robust upregulation of CELF2 and PABPC5 , both of are associated with expression of distal PAS and may therefore contribute to the 3´UTR lengthening observed in this study ( 64 , 76 , 77 ). Howe v er, some differentially expressed genes associated with polyadenylation did not change in a manner consistent with recent findings. A major example is PABPN1 , a gene strongly associated with polyadenylation efficiency b y tethering cleav age and poly adenylation specificity factor (CPSF) with pol y(A) pol ymerase during pol y(A) tail extension ( 78 ). This effector also promotes usage of distal PAS by binding proximal PAS and pre v enting their cleavage ( 76 , 78 , 79 ). Despite these findings, significant downregulation of CPSF was observed during differentiation, suggesting if this gene contributes to the patterns of 3´UTR and poly(A) tail e xtension observ ed during neuronal differentia tion, its functional sta tus circumv ents its mRNA le vels. Likewise, no significant changes were observed f or an y pol y(A) pol ymerase genes, suggesting poly(A) tail length modulation was not a consequence of altered pol y(A) pol ymerase expression.
In summary, our study suggests that mRNA 3´UTR and poly(A) tail length regulation is prevalent during neuronal dif ferentia tion and exhibits varying degrees of association with mRNA expression, translation and miRNA. Since 3ḿ RNA features encode significant regulatory capacity, we suspect alternati v e polyadenylation and poly(A) tail length modulation contribute substantially to the unique spatial and temporal distribution of gene expression in neurons.
Howe v er, further mechanistic analysis of these systems will be r equir ed to fully elucidate their functional significance in the neuronal context under normal and pathological conditions.

DA T A A V AILABILITY
All raw and processed sequencing data are currently available online at the Gene Expression Omnibus (GEO), accession number GSE155432.

SUPPLEMENT ARY DA T A
Supplementary Data are available at NAR Online.