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Analysis and design of RNA sequencing experiments for identifying mRNA isoform regulation

ABSTRACT: Through alternative splicing, most human genes express multiple isoforms that may have distinct or even antagonistic functions. To infer isoform regulation based on data from high-throughput sequencing of cDNA fragments (RNA-Seq), we have developed MISO, a computational model that estimates the expression level of alternatively spliced exons and mRNA isoforms and provides intuitive measures of conﬁdence in these estimates. Incorporation of the length distribution of inserted cDNA fragments in paired-end RNA-Seq analysis in MISO enables dramatic improvements in estimation of alternative splicing levels relative to previous methods. We show that one lane of paired-end RNA-Seq data can provide far more information about splicing than two lanes of single-end data, depending critically on properties of the distribution of cDNA fragment lengths in the sequenced library. MISO also leads to an intuitive method to detect diﬀerentially regulated exons or isoforms. Application of this method implicates the RNA splicing factor hnRNP H in regulation of alternative cleavage and polyadenylation, a role that is supported by UV crosslinking/immunoprecipitation/high-throughput sequencing (CLIP-Seq) analysis. Together, our results provide a probabilistic framework for RNA-Seq analysis, derive functional insights into pre-mRNA processing, and yield guidelines for the optimal design of RNA-Seq experiments for studies of gene and isoform expression.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE23694 | GEO | 2010/11/07

SECONDARY ACCESSION(S): PRJNA130865

REPOSITORIES: GEO

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Analysis and design of RNA sequencing experiments for identifying mRNA isoform regulation

Project description:Through alternative splicing, most human genes express multiple isoforms that may have distinct or even antagonistic functions. To infer isoform regulation based on data from high-throughput sequencing of cDNA fragments (RNA-Seq), we have developed MISO, a computational model that estimates the expression level of alternatively spliced exons and mRNA isoforms and provides intuitive measures of con?dence in these estimates. Incorporation of the length distribution of inserted cDNA fragments in paired-end RNA-Seq analysis in MISO enables dramatic improvements in estimation of alternative splicing levels relative to previous methods. We show that one lane of paired-end RNA-Seq data can provide far more information about splicing than two lanes of single-end data, depending critically on properties of the distribution of cDNA fragment lengths in the sequenced library. MISO also leads to an intuitive method to detect di?erentially regulated exons or isoforms. Application of this method implicates the RNA splicing factor hnRNP H in regulation of alternative cleavage and polyadenylation, a role that is supported by UV crosslinking/immunoprecipitation/high-throughput sequencing (CLIP-Seq) analysis. Together, our results provide a probabilistic framework for RNA-Seq analysis, derive functional insights into pre-mRNA processing, and yield guidelines for the optimal design of RNA-Seq experiments for studies of gene and isoform expression. CLIPseq of hnRNP H in HEK 293T cells. RNAseq of polyA+ RNA from C2C12 mouse myoblasts stably expressing an empty vector or a vector containing an shRNA against CUGBP1. Libraries of two different insert lengths were created and examined.

2010-11-07 | E-GEOD-23694 | biostudies-arrayexpress

Alternative splicing regulation by homologous Muscleblind proteins

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Alternative splicing regulation by homologous Muscleblind proteins

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2016-06-01 | E-GEOD-79095 | biostudies-arrayexpress

Identification of the O-GlcNAc-regulated alternative splicing events by performing RNA sequencing on HeLa cells with altered O-GlcNAc level.

Project description:We performed RNA sequencing on HeLa cells treated with OGT inhibitor (Ac4-5SGlcNAc), DMSO, or OGA inhibitor (Thiamet-G) and obtained about 100 million paired-end reads (150 bp) for each replicate. Then the sequencing data was analyzed by MISO to select the O-GlcNAc-regulated alternative splicing events.

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Translational profiling of RBMX-overexpressed HEK293 cells

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Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing

Project description:Alternative pre-mRNA splicing has long been proposed to greatly contribute to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom spectral library trained on analysis of RNA-Seq data with MAJIQ, an algorithm optimized to detect differential and unannotated junction usage for a given splice site. Using this custom library to match against tandem mass spectra acquired by data independent acquisition (DIA), we improved identification of splicing-derived proteoforms by ~30% as compared to use of the SwissProt database alone. Moreover, our increased depth and detection of proteins allowed us to track changes in the transcriptome and proteome induced by T cell stimulation, as well as fluctuations in protein sub-cellular localization. In sum, our data here confirms that use of generic databases in proteomic studies under-estimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

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