Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing

Hoque, Mainul; Ji, Zhe; Zheng, Dinghai; Luo, Wenting; Li, Wencheng; You, Bo; Park, Ji Yeon; Yehia, Ghassan; Tian, Bin

doi:10.1038/nmeth.2288

Cited by 381 publications

(498 citation statements)

References 42 publications

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“…(B) Aggregation of the location of each aligned read, using the annotated transcription start sites (TSSs) and transcription termination sites (TTSs) as reference, for 5 ′ (green) and 3 ′ libraries (red). Shepard et al 2011;Derti et al 2012;Hoque et al 2013). Taken together, these observations suggest that a robust end-sequence quantification method must find transcript ends de novo, rather than rely on gene annotations, as well as filter out any read alignments to internal polyadenylation sites.…”

Section: Accurate Mapping Of End-sequence Libraries Presents Unique Cmentioning

confidence: 99%

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

et al. 2016

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RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared endsequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3 ′ -isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct β-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.

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Section: Accurate Mapping Of End-sequence Libraries Presents Unique Cmentioning

confidence: 99%

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

et al. 2016

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“…22 APA isoforms generally differ in their 3′UTR, but in about one third of the cases, they also differ in coding sequences. 23 Because the 3′UTR generally harbors functional domains such as microRNA or RBP-binding sites, altered 3′UTR length by APA may have profound effects on gene expression. 24 Alternative first exon usage occurs when the transcriptional machinery starts at a different promoter and leads to different mRNA or protein isoforms at the N terminus.…”

Section: Different Types Of Alternative Splicingmentioning

confidence: 99%

RNA Splicing

2016

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RNA splicing, a post-transcriptional process necessary to form a mature mRNA, was discovered in the late 1970s. 1 Two different modes of splicing have been defined, that is, constitutive splicing and alternative splicing. Constitutive splicing is the process of removing introns from the premRNA, and joining the exons together to form a mature mRNA. Alternative splicing, on the other hand, is the process where exons can be included or excluded in different combinations to create a diverse array of mRNA transcripts from a single pre-mRNA and therefore serves as a process to increase the diversity of the transcriptome. The estimated number of alternative splicing events in the human transcriptome has risen sharply over the past decades. In the 1980s, it was thought that about 5% of human genes were subjected to alternative splicing.2 In 2002, this number had risen to 60%, 3 and now, after implementation of next-generation-sequencing technologies, we know that the vast majority, >95% of mRNAs, are subjected to alternative splicing. 4 Nevertheless, the function of a large fraction of these splice isoforms remains to be elucidated. Furthermore, it is anticipated that in different tissues, or in tissues with different disease states, new isoforms still remain to be identified. 5The process of splicing is highly conserved during evolution. Splicing is more prevalent in multicellular than in unicellular eukaryotes because of the lower number of introncontaining genes in the latter. 6 Later in evolution, alternative splicing becomes more prevalent in vertebrates than in invertebrates. Interestingly, just a single exon-skipping event in the RNA-binding protein (RBP), polypyrimidine tract binding protein 1 has been shown to direct numerous alternative splicing changes between species, indicating that a single splicing event can amplify transcriptome diversity between species. 7The recent observation that the total number of protein-coding genes does not differ much between species, fueled the hypothesis that alternative splicing largely contributes to organism diversity. And indeed, as we move up the phylogenetic tree, alternative splicing complexity increases, with the highest complexity in primates. 8,9The aim of this review is to give a comprehensive overview of all aspects of constitutive and alternative splicing and their regulators, as well as the importance of these different aspects in human disease, with a focus on the heart. In addition, we discuss possible therapeutic interventions and try to uncover potential future directions of research. Major and Minor SpliceosomeRNA splicing is performed by the spliceosome, a large and dynamic ribonucleoprotein complex composed of proteins and small nuclear RNAs (snRNAs), which assembles on the pre-mRNA (Figure 1). Ribonucleoproteins consist of 1 or 2 small nuclear RNAs (snRNAs; U1, U2, U4/U6, or U5), and a variable number of complex-specific proteins.

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“…Therefore, ApA provides an additional layer of complexity in regulating gene expression at the posttranscriptional level. Powerful high-profiling technologies focusing on 3 0 -UTRs of mRNAs provided high-resolution snap shots of alternatively polyadenylated mRNA isoforms in various tissues and cells across many species [6][7][8][9][10][11] . An important insight that emerged from these studies is that 3 0 -UTR length undergoes dynamic changes under pathogenic conditions such as cancer and in diverse biological processes such as cell proliferation, differentiation and development 2,6,7,[12][13][14] .…”

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confidence: 99%