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Alternative 3’UTRs play a widespread role in translation-independent mRNA association with endoplasmic reticulum

ABSTRACT: Transcripts encoding membrane and secreted proteins are known to undergo translation on endoplasmic reticulum (ER). Translation-independent ER association (TiERA) has been reported for certain mRNAs, but the phenomenon is poorly understood. Here, using cell fractionation, polysome profiling, and 3’ end sequencing, we examine TiERA of poly(A)+ RNAs in mouse C2C12 myoblast cells. We identify transcript features that facilitate TiERA, including transcript size and GG and GC content. Consistent with the feature analysis, alternative polyadenylation (APA) isoforms differ substantially in TiERA, with long 3’UTR isoforms generally having a higher TiERA potential than short 3’UTR isoforms. Importantly, the widespread 3’UTR lengthening taking place in cell differentiation leads to greater transcript association with ER in differentiated myotubes, despite that the TiERA potential being generally maintained. In addition, we show that TiERA correlates negatively with mRNA stability, highlighting 3’UTR-mediated mRNA decay on ER. Together, our data indicate that sequence and size features impact ER association independent of translation, leading to distinct mRNA metabolism for different transcript groups, and APA can alter transcript stability and translation through isoform-specific ER association.

ORGANISM(S): Mus musculus

PROVIDER: GSE162971 | GEO | 2021/07/20

REPOSITORIES: GEO

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Alternative 3’UTRs play a widespread role in mRNA association with the endomembrane system

Project description:Transcripts encoding membrane and secreted proteins are known to have translation on endoplasmic reticulum (ER) through the signal recognition particle (SRP)-mediated mechanism. Translation-independent association of mRNAs with ER has also been reported, but poorly understood. Here, using 3’ end sequencing, we examine transcripts associated with polysomes on ER in mouse C2C12 cells. We identify transcript features that facilitate translation-independent ER association, including CDS size, 3’UTR size, and GC content. Interestingly, transcripts encoding nuclear proteins tend to have a higher ER association potential than other transcripts, suggesting localized translation to facilitate nuclear import. We show long 3’UTR isoforms are generally more likely to be associated with ER than short isoforms. Consistently, global shortening of 3’UTRs by knocking down CFI-25 expression leads to substantial alternation of mRNA association with ER. While ER association potentials of transcripts undifferentiated myoblasts are similar to those in differentiated myotubes, 3’UTR lengthening in differentiation leads to more transcripts associated with ER in myotubes. Moreover, translation-independent ER association correlates with low mRNA stability, which is exacerbated in cells with oxidative stress. Together, our data indicate that sequence and structure features of mRNAs facilitate ER association and translation, potentially impacting localization dynamics of protein products, and cellular stress remodels membrane-associated transcripts through mRNA stability regulation.

2023-04-15 | GSE150607 | GEO

Comparative analysis of multiple human cell lines reveals distinct stability regulation of alternative polyadenylation isoforms and mitochondrial RNAs in neuronal cells

Project description:mRNA stability plays an important role in gene expression. Here, using 3’ end sequencing of newly made and steady-state poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having high GC content and likely more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to other two. These features also differentiate alternative polyadenylation (APA) 3’UTR isoforms in their stability difference in a cell type-specific manner. By analyzing cell differentiation of a neural stem cell line, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirms the neuronal identity of SH-SY5Y cells at gene expression and APA levels. Through comparison of APA isoforms, we show that mRNA stability control buffer the cleavage and polyadenylation site (PAS) choice in the cells studied. In addition, transcripts using intronic PAS are generally less stable, especially when the intron harboring the PAS is large and has a strong 5’ splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Moreover, we found that poly(A)+ RNAs encoded by the mitochondrial genome are more stable in SH-SY5Y cells than the other two cell types, suggesting that mitochondrial RNA metabolism is distinct in neurons. Together, our results reveal multiple cell-specific mechanisms by which mRNA stability could contribute to the gene expression and APA programs that are important for cell identity.

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Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products

Project description:Alternative polyadenylation (APA) produces transcript 3’ untranslated regions (3’UTRs) with distinct sequences, lengths, stability, and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3’UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3’UTR products of APA, leading to their systematic downregulation. Further, we find that many APA events consistently observed in multiple tumor types are controlled by NMD. Additionally, PTBP1, previously implicated in direct modulation of co-transcriptional polyA site choice, regulates the balance of short and long 3’UTR isoforms by inhibiting NMD. Our data suggest that PTBP1 binding near polyA sites can drive production of long 3’UTR APA products in the nucleus and/or protect them from decay in the cytoplasm. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

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Alternative cleavage and polyadenylation in spermatogenesis coordinates chromatin regulation and post-transcriptional control

Project description:Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3âUTRs in testes. Here we show widespread shortening of 3âUTR by APA during the first wave of spermatogenesis in mouse, with 3âUTRs being the shortest in spermatids. Shortening of 3âUTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear to be highly potent for transcript elimination during spermatogenesis. We additionally found widespread regulation of APA in introns and global activation of upstream antisense transcripts during spermatogenesis. Interestingly, genes that display 3âUTR shortening tend to have higher levels of H3K4me3, consistent with the open chromatin feature previously observed in spermatids. Since genes with 3âUTR shortening tend to have functions important for further sperm development after spermatids, when transcription is halted, this result indicates that expression of short, stable mRNAs may serve the purpose of mRNA storage for later translation. Thus, APA in spermatogenesis connects regulation of chromatin status with post-transcriptional control, and impacts sperm maturation. 3'READS of 1 week to 6 week of testis development

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A compendium of conserved cleavage and polyadenylation events in mammalian genes

Project description:Cleavage and polyadenylation is essential for 3’ end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3’UTRs and/or coding sequences. Here we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on ~1.2 billion 3’ end sequencing reads from over 360 human, mouse and rat samples. We show that ~80% of mammalian mRNA genes contain at least one conserved PAS, and ~50% have conserved APA events. PAS conservation generally reduces promiscuous 3’ end processing, stabling gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with APA events. While tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. We show enrichment of mRNA destabilizing motifs in alternative 3’UTR sequences, leading to substantial differences in mRNA stability between 3’UTR APA isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Mutations of the U-rich motif around the PAS often accompany APA profile changes between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation involving evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic understanding of APA functions.

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The mRNA export receptor NXF1 coordinates transcriptional dynamics, alternative polyadenylation and mRNA export

Project description:Alternative polyadenylation (APA) produces mRNA isoforms with different 3’UTR lengths. Previous studied indicated that 3’ end processing and mRNA nuclear export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to expression of long 3’UTR isoforms. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal a number of gene features that impact NXF1-dependent APA, including 3’UTR size, gene size and AT content. Surprisingly, downregulation of NXF1 results in accumulation of RNAP II at the 3’ end of genes, correlating with its role in APA regulation. Moreover, we show that NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3’UTR isoform with UGUA motifs. Together, our work reveals several important roles of NXF1 in coordinating RNAPII distribution, 3’ end processing, and mRNA export of long 3’UTR transcripts, implicating NXF1 as the nexus of gene expression regulation.

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Cellular stress alters 3’UTR landscape through alternative polyadenylation and isoform-specific degradation

Project description:Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different lengths of 3’ untranslated region (3’UTR). Here we show arsenic stress elicits global shortening of 3’UTRs through two mechanisms. First, stress leads to immediate shortening of 3’UTR due to preferential usage of proximal cleavage and polyadenylation sites (PASs), as revealed by 3’ end sequencing of newly made RNAs that are metabolically labeled with 4-thiouridine. Second, long 3’UTR isoforms are more rapidly degraded during recovery from stress as compared to short 3’UTR isoforms, further shortening 3’UTR lengths in the cell. Using ribonucleoprotein immunoprecipitation coupled with 3’ end sequencing (3’READS+RIP), we show that the RNA-binding protein T cell-restricted intracellular antigen-1 (Tia1) preferentially interacts with long 3’UTR isoforms via U-rich elements in alternative 3’UTR sequences, and the interaction correlates with stress granule (SG) association during stress and with mRNA decay during recovery from stress, indicating SG-mediated RNA clearance mechanism post stress. Importantly, genes whose 3’UTRs are shortened by APA during stress can evade stress-induced 3’UTR size-based mRNA degradation, leading to higher transcript abundance post stress. Moreover, proliferating and differentiated cells display different extents of 3’UTR shortening after stress, indicating cell type-specific of impact of stress on the 3’UTR landscape. Together, our data indicate that 3’UTR length plays important roles in gene expression in stressed cells, and APA functions as an adaptive stress response mechanism to preserve mRNAs.

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Alternative polyadenylation alters protein dosage by switching between intronic and 3' UTR sites

Project description:Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3' UTR, introns, or exons. Most studies focus on APA within the 3' UTR, but here we show that CPSF6 insufficiency alters protein levels and causes a developmental syndrome by deregulating APA throughout the transcript. In neonatal humans and zebrafish larvae, CPSF6 insufficiency shifts poly(A) site usage between the 3'UTR and internal sites in a pathway-specific manner. Genes associated with neuronal function undergo mostly intronic APA, reducing their expression, while genes associated with heart and skeletal function mostly undergo 3' UTR APA and are upregulated. This suggests that, in healthy conditions, cells toggle between internal and 3'UTR APA to modulate protein expression.

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