Alternative polyadenylation (APA) affects most mammalian genes. The genome-wide investigation of APA has been hampered by an inability to reliably profile it using conventional RNA-seq. We describe ...'Quantification of APA' (QAPA), a method that infers APA from conventional RNA-seq data. QAPA is faster and more sensitive than other methods. Application of QAPA reveals discrete, temporally coordinated APA programs during neurogenesis and that there is little overlap between genes regulated by alternative splicing and those by APA. Modeling of these data uncovers an APA sequence code. QAPA thus enables the discovery and characterization of programs of regulated APA using conventional RNA-seq.
Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these ...goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Previous transcriptomic profiling studies have typically focused on separately analyzing mRNA expression, alternative splicing and alternative polyadenylation differences between cell and tissue ...types. However, the relative contribution of these three transcriptomic regulatory layers to cell type specification is poorly understood. This question is particularly relevant to neurons, given their extensive heterogeneity associated with brain location, morphology and function. In the present study, we generated profiles for the three regulatory layers from developmentally and regionally distinct subpopulations of neurons from the mouse hippocampus and broader nervous system. Multi-omics factor analyses revealed differing contributions of each transcriptomic layer in the discrimination of neurons based on their stage of development, region, and function. Importantly, profiles of differential alternative splicing and polyadenylation better discriminated specific neuronal subtype populations than gene expression patterns. These results provide evidence for differential relative contributions of coordinated gene regulatory layers in the specification of neuronal subtypes.
Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to ...be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.
Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity and is frequently misregulated in human disease. Accordingly, an important goal ...of biomedical research is the development of tools capable of comprehensively, accurately, and efficiently profiling AS. Here, we describe Whippet, an easy-to-use RNA-seq analysis method that rapidly—with hardware requirements compatible with a laptop—models and quantifies AS events of any complexity without loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that one-third of human protein coding genes produce transcripts with complex AS events involving co-expression of two or more principal splice isoforms. We observe that high-entropy AS events are more prevalent in tumor relative to matched normal tissues and correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate future biomedical research.
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•Whippet, a new method for the rapid and accurate profiling of alternative splicing•Whippet reliably detects and quantifies complex alternative splicing events•Approximately one-third of human genes simultaneously express multiple major isoforms•Complex splicing events are conserved, tissue regulated, and more prevalent in cancer
Sterne-Weiler et al. describe Whippet, a method for the rapid and accurate quantitative profiling of alternative splicing from RNA-seq data. Whippet is particularly effective in the analysis of complex alternative splicing events, revealing that they impact up to 40% of human genes, are often conserved, and are elevated in cancer.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Global transcriptomic imbalance is a ubiquitous feature associated with cancer, including hepatocellular carcinoma (HCC). Analyses of 1,225 clinical HCC samples revealed that a large numbers of RNA ...binding proteins (RBPs) are dysregulated and that RBP dysregulation is associated with poor prognosis. We further identified that oncogenic activation of a top candidate RBP, negative elongation factor E (NELFE), via somatic copy-number alterations enhanced MYC signaling and promoted HCC progression. Interestingly, NELFE induces a unique tumor transcriptome by selectively regulating MYC-associated genes. Thus, our results revealed NELFE as an oncogenic protein that may contribute to transcriptome imbalance in HCC through the regulation of MYC signaling.
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•mRNA binding proteins (RBPs) are globally dysregulated in HCC•Tumor-associated RBPs are predictive of HCC patient survival•NELFE is oncogenic and enhances MYC signaling•NELFE regulates MYC signaling by binding directly to MYC or its targets
Dang et al. show that a large numbers of RNA binding proteins (RBPs) are dysregulated in hepatocellular carcinoma (HCC) and that NELFE, an RBP, enhances MYC-induced HCC development by regulating the binding of MYC to target promoters and the mRNA stability of several MYC-regulated genes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and ...regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.
Expression levels of many human genes are under the genetic control of expression quantitative trait loci (eQTLs). Despite technological advances, the precise molecular mechanisms underlying most ...eQTLs remain elusive. Here, we use deep mRNA sequencing of two CEU individuals to investigate those mechanisms, with particular focus on the role of splicing control loci (sQTLs). We identify a large number of genes that are differentially spliced between the two samples and associate many of those differences with nearby single nucleotide polymorphisms (SNPs). Subsequently, we investigate the potential effect of splicing SNPs on eQTL control in general. We find a significant enrichment of alternative splicing (AS) events within a set of highly confident eQTL targets discovered in previous studies, suggesting a role of AS in regulating overall gene expression levels. Next, we demonstrate high correlation between the levels of mature (exonic) and unprocessed (intronic) RNA, implying that ∼75% of eQTL target variance can be explained by control at the level of transcription, but that the remaining 25% may be regulated co- or post-transcriptionally. We focus on eQTL targets with discordant mRNA and pre-mRNA expression patterns and use four examples: USMG5, MMAB, MRPL43, and OAS1, to dissect the exact downstream effects of the associated genetic variants.
Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively ...sequencing complete coding regions (i.e., "whole exome") have the potential to contribute our understanding of human diseases. We used a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular-receptor-family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point-mutations, insertions-deletions, and intronic splice-site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole-exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity. Hum Mutat 31:1-6, 2010.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We ...describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large “missing cache” of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.
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•SPAR-seq is a versatile method for elucidating endogenous RNA regulatory networks•Hundreds of trans-acting factors control ES and neural splicing networks•SPAR-seq defines positive and negative functional and physical interrelationships•Transcription factors have dual direct and indirect roles in splicing regulation
Han, Braunschweig et al. describe SPAR-seq, a strategy for the systematic elucidation of endogenous RNA regulatory networks. Hundreds of factors controlling cell-fate-associated splicing networks are defined, among which are transcription factors that have dual direct and indirect regulatory roles.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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