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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

2016-06-01 | GSE79095 | GEO

Alternative splicing regulation by homologous Muscleblind proteins

Project description:We provide data showing alternative splicing regulation by Muscleblind proteins in MEFs. MEFs lacking functional Muscleblind (DKO MEFs) were stably reconstituted with Muscleblind proteins from Homo sapiens, Ciona intestinalis, Drosophila melanogaster, Caenorhabditis elegans or Trichoplax adhaerens and splicing regulation was explored using RNA-seq analysis followed by MISO (Mixture of Isoforms). Alternative splicing was accessed using RNA-sequencing data from five DKO MEF lines reconstituted with different GFP-tagged Muscleblind homologs or GFP alone and compared to RNA-seq data from three WT MEF lines and three control DKO MEFs (no Muscleblind reconstitution). A total of 12 samples were used for high-throughput sequencing.

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.

2019-05-13 | GSE131067 | GEO

Translational profiling of RBMX-overexpressed HEK293 cells

Project description:Wild-type and RBMX-overexpressed HEK293 cells where profiled at the transcriptome and translatome levels through polysomal profiling. Paired-end RNA-seq libraries were built and sequenced on an Illumina HiSeq 2000 machine to allow for accurate gene expression quantification and alternative splicing isoforms detection.

2019-12-01 | GSE68990 | GEO

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.

2019-10-14 | PXD012556 | Pride

Developmental arrest of Drosophila survival motor neuron (Smn) mutants accounts for differences in expression of minor intron-containing genes

Project description:RNA-seq was used to analyze mRNA levels and splicing differences between wild-type (Oregon R) and Smn null mutant larvae. Illumina RNA-seq of Oregon R and Smn mutant age-matched larvae from RNA extracted on day four post egg laying. Conclusions from this study used the publicly available modENCODE data from Graveley et al. Nature 2011 (in addition to our RNA-seq data). We downloaded their SRA files, dumped them to fastq files, and put them through our same analysis pipelines. The renalysis data and details are provided in 'reanalysis.tar' and readme_reanalysis.txt. The Cufflinks processed file outputs (22 files in 'Cufflinks_outputs.tar') are derived from all of our raw sample files and all of the developmental data mentioned in the readme.txt file. The file format and content description is provided in the 'readme_for_Cufflinks.txt' Unlike Cufflinks cuffdiff analysis that merged our data with the developmental data, the MISO and DEXSeq analysis was carried out in parallel with analysis of our samples. This generated two additional files for the DEXSeq developmental analysis (DEXSeq output; paired end developmental Graveley et al. Nature 2011): L2_L3P1_2_DEUtable.txt L312hr_L3P1_2_DEUtable.txt Please note that MISO lacks the capacity to incorporate replicates, which contributed to the abundance of these file types. MISO output file names are prepended with the respective sample/raw data names. For example, EG003_EG007.*miso_bf.txt.gz are MISO comparison of Ore-R R1 (EG003) and Smn R1 (EG007).

2013-08-07 | E-GEOD-49587 | biostudies-arrayexpress

Aberrant splicing isoforms detected by full-length transcriptome sequencing as transcripts of potential neoantigens in non-small cell lung cancer

Project description:To identify aberrant splicing isoforms and potential neoantigens, we performed full-length cDNA sequencing of lung adenocarcinoma cell lines using a long-read sequencer MinION. We constructed a comprehensive catalog of aberrant splicing isoforms and detected isoform-specific peptides using proteome analysis.

2020-12-09 | PXD019915 | JPOST Repository

Transcriptome-wide analysis of wild-type and SRSF3-knockout glioma stem-like cells

Project description:Purpose: The splicing factor SRSF3 is a member of serine- and arginine-rich proteins, which is frequently upregulated in various types of cancer. The aim of this study is to profile the alternative splicing (AS) events that were regulated by SRSF3 in glioma stem-like cells (GSCs). Methods: Total RNAs isolated from GSC83 and GSC528 cells with SRSF3-konckout (KO) or control (WT) were subjected to paired-end RNA-seq using the Illumina NextSeq 500 system according to the manufacturer's instruction. Raw reads were subjected to quality control and then aligned with HISAT2 to the human genome hg19. Quantification was performed using HTSeq-count. Differential expression of genes was calculated by DESeq2. For the splicing analysis, the mapped reads were processed through the MISO pipeline to estimate expression of alternatively spliced isoforms. The PSI (Percent Spliced In) values were calculated for each annotated AS event. Results: We identified a total of 2752 and 2548 SRSF3-regulated AS events in GSC83 and 528 cells, respectively, with 1143 events shared by these two GSC cell lines. Conclusions: Our study defines SRSF3 as a master regulator of glioma-associated AS program, implicating SRSF3 as an oncogenic factor for tumorigenesis and a potential therapeutic target for GBM treatment.

2019-09-21 | GSE130501 | GEO

RNA-Seq analysis of Arabidopsis thaliana splicing factor AtU2AF65a and AtU2AF65b against the wild type (Col).

Project description:Two Arabidopsis thaliana splicing factor [AtU2AF65 isoforms (AtU2AF65a and AtU2AF65b)] mutants displayed the opposite flowering phenotypes. To assay the RNA processing including alternative splicing and pre-mRNA splicing of target genes of this protein in Arabidopsis, the 7-day seedlings (shoot apices) of wild type atu2af65a and atu2af65b mutants were used for RNA-Seq.

2023-05-01 | E-MTAB-12648 | biostudies-arrayexpress

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