Regulation of cleavage and polyadenylation (CPA) affects gene expression and polyadenylation site (PAS) choice. Here, we report that the CPA and termination factor PCF11 modulates gene expression on ...the basis of gene size. Although downregulation of PCF11 leads to inhibition of short gene expression, long genes are upregulated because of suppressed intronic polyadenylation (IPA) enriched in large introns. We show that this regulatory scheme, named PCF11-mediated expression regulation through IPA (PEIPA), takes place in cell differentiation, during which downregulation of PCF11 is coupled with upregulation of long genes with functions in cell morphology, adhesion, and migration. PEIPA targets distinct gene sets in different cell contexts with similar rules. Furthermore, PCF11 is autoregulated through a conserved IPA site, the removal of which leads to global activation of PASs close to gene promotors. Therefore, PCF11 uses distinct mechanisms to regulate genes of different sizes, and its autoregulation maintains homeostasis of PAS usage in the cell.
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•PCF11 regulates mRNA expression on the basis of gene size•Genes with large introns are regulated by PCF11 through intronic polyadenylation•PCF11 controls its own gene expression through an intronic polyadenylation site•Long gene and PCF11 expression levels are inversely correlated globally
Wang et al. report a gene size-based dichotomic scheme by which the cleavage and polyadenylation factor PCF11 modulates gene expression. Distinct polyA site types are involved to regulate short and long genes. PCF11 expression level is autoregulated and is connected with long gene expression in cell differentiation and across tissues.
Sequencing of the 3′ end of poly(A)
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RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3′ region extraction and deep ...sequencing (3′READS), to address mispriming issues that often plague 3′ end sequencing. Here we report a new version, named 3′READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)
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RNA and to remove the bulk of the poly(A) tail, 3′READS+ generates RNA fragments with an optimal number of terminal A's that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3′READS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (∼50%). 3′READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3′ end counting, especially when the amount of input total RNA is limited.
Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3' region extraction and deep sequencing (3'READS) to address mispriming issues that commonly plague ...poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3' ends revealed over 5,000 previously overlooked pAs (∼8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3'READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3' end-processing activity in development and differentiation.
Abstract
Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different 3′UTR lengths, production of which is influenced by cellular conditions. Here, we show that arsenic ...stress elicits global shortening of 3′UTRs through preferential usage of proximal polyadenylation sites during stress and enhanced degradation of long 3′UTR isoforms during recovery. We demonstrate that RNA-binding protein TIA1 preferentially interacts with alternative 3′UTR sequences through U-rich motifs, correlating with stress granule association and mRNA decay of long 3′UTR isoforms. By contrast, genes with shortened 3′UTRs due to stress-induced APA can evade mRNA clearance and maintain transcript abundance post stress. Furthermore, we show that stress causes distinct 3′UTR size changes in proliferating and differentiated cells, highlighting its context-specific impacts on the 3′UTR landscape. Together, our data reveal a global, 3′UTR-based mRNA stability control in stressed cells and indicate that APA can function as an adaptive mechanism to preserve mRNAs in response to stress.
Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type ...critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.
Most eukaryotic genes express alternative polyadenylation (APA) isoforms. A growing number of RNA sequencing methods, especially those used for single-cell transcriptome analysis, generate reads ...close to the polyadenylation site (PAS), termed nearSite reads, hence inherently containing information about APA isoform abundance. Here, we present a probabilistic model-based method named MAAPER to utilize nearSite reads for APA analysis. MAAPER predicts PASs with high accuracy and sensitivity and examines different types of APA events with robust statistics. We show MAAPER's performance with both bulk and single-cell data and its applicability in unpaired or paired experimental designs.
Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3' untranslated regions (3'UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors ...regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.
Alternative polyadenylation (APA) produces mRNA isoforms with different 3′ UTR lengths. Previous studies indicated that 3′ end processing and mRNA 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 long 3′ UTR isoform expression. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal several gene features that impact NXF1-dependent APA, including 3′ UTR size, gene size, and AT content. Surprisingly, NXF1 downregulation results in RNA polymerase II (Pol II) accumulation at the 3′ end of genes, correlating with its role in APA regulation. Moreover, NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3′ UTR isoform with UGUA motifs. Together, our work reveals important roles of NXF1 in coordinating transcriptional dynamics, 3′ end processing, and nuclear export of long 3′ UTR transcripts, implicating NXF1 as a nexus of gene regulation.
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•mRNA export factors generally promote expression of long 3′ UTR isoforms•Gene size and AT content impact alternative polyadenylation of 3′ UTRs•NXF1 regulates RNA polymerase II distribution along the gene•NXF1 together with CFI-68 facilitates nuclear export of long 3′ UTR transcripts
3′ end processing and mRNA export are intertwined in gene regulation. Chen et al. show that the mRNA export receptor NXF1 globally impacts Pol II dynamics, enhances expression of long 3′ UTR isoforms, and facilitates their nuclear export, implicating NXF1 as a nexus for gene expression.
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain ...largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.
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