Analysis of alternative cleavage and polyadenylation by 3' region extraction and deep sequencing
Ontology highlight
ABSTRACT: 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' endM-bM-^@M-^Sprocessing activity in development and differentiation. 3'READS to map pAs in mouse genome
Project description:Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. We first analyzed HeLa cell transcriptome using PAS-Seq to demonstrate that PAS-Seq not only accurately and comprehensively identifies poly(A) junctions, but also provide quantitative information on the relativel abundance of APA isoforms. Next we analyzed the mouse embryonic stem cells, neural stem/progenitor cells, and neurons by PAS-Seq to characterize the dynamic changes of mRNA polyadenylation during stem cell differentiation.
Project description:Sequencing of the 3â end of poly(A)+ RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3â region extraction and deep sequencing (3âREADS), to address mispriming issues that often plague 3â end sequencing. Here we report a new version, named 3âREADS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)+ RNA and to remove bulk of the poly(A) tail, 3âREADS+ generates RNA fragments with an optimal number of terminal As 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 3âREADS+, 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%). 3âREADS+ will be a useful tool to accurately study APA and to analyze gene expression by 3â end counting, especially when the amount of input total RNA is limited. Nine 3'READS+ libraries were made with different amounts (100 ng, 200 ng, 400 ng, 1000 ng, 5000 ng, 15000 ng) of input Hela RNA.
Project description:Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage and 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 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 influencing C/P events in 5’ introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, 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 indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells
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
Project description:SMORE-seq (Simultaneous Mapping Of RNA Ends by sequencing) is developted to simultaneously identify the strongest TSS (Transcription Start Site) and PAS (Polyadenylation Site) SMORE-seq were performed in wild type Saccharomyces cerevisiae
Project description:Both canonical and alternative splicing of RNAs is governed by intronic sequence elements and produces transient lariat structures fastened by branch-points within introns. To map precisely the location of branch-points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes, and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon skipping branch-points. LaSSO thus provides an effective, accurate and unbiased approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated to RNA-seq pipelines. Interrogation of the S. pombe transcriptome using rRNA depleted strand specific RNA sequencing (Illumina HiSeq 2000) in wild type and dbr1.M-NM-^T cultures. A total of 4 samples were analyzed: two biological repeates of wild-type strain and two biological repeats of dbr1.M-NM-^T
Project description:Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage and 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 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 influencing C/P events in 5’ introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, 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 indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions.
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.
Project description:Differences in gene regulation contribute to phenotypic differences between humans and other primates. While co-transcriptional gene regulatory mechanisms such as alternative polyadenylation (APA) can help explain how variation in gene regulation manifests, such mechanisms remain understudied. We measured polyadenylation site (PAS) usage in a panel of 6 human and 6 chimpanzee lymphoblastoid cell lines (LCLs). While APA is largely conserved between humans and chimpanzees, genes with divergent APA patterns are enriched among differentially expressed and differentially translated genes. Differential usage of 3’ UTR and intronic PAS are both significantly correlated with differential mRNA expression effect sizes but in opposite directions. For many genes, there is one PAS dominant, meaning it is used much more often than others. The dominant PAS for these gene is overwhelmingly shared between species, however, differences in dominant PAS are enriched for genes with expression differences. Finally, through post-translational mechanisms, we believe APA contributes to genes differentially expressed at the protein level but not in mRNA. As this is the first primate comparative study of APA, our study establishes APA as a key mechanism underlying the genetic regulation of gene and protein expression levels in primates.
Project description:The aim of this study was to identify target genes of the SR protein kinase SPK-1. spk-1(RNAi) leads to defects in cell polarity. We were therefore primarily interested in genes who exhibit changes in splicing regulation upon depletion of SPK-1 and could underlie the polarity phenotype of spk-1(RNAi). Transcriptome profiling of control and spk-1(RNAi) worms by RNAseq