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Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing

ABSTRACT: We carried out the first analysis of alternative splicing complexity in human tissues using mRNA-Seq data. New splice junctions were detected in 20% of multiexon genes, many of which are tissue specific. By combining mRNA-Seq and EST-cDNA sequence data, we estimate that transcripts from 95% of multiexon genes undergo alternative splicing and that there are 100,000 intermediate- to high-abundance alternative splicing events in major human tissues. From a comparison with quantitative alternative splicing microarray profiling data, we also show that mRNA-Seq data provide reliable measurements for exon inclusion levels. Keywords: mRNA expression 32-nucleotide sequence reads from six human tissues including brain, cerebral cortex, heart, liver, lung and skeletal muscle.

ORGANISM(S): Homo sapiens

SUBMITTER: Weijun Gao

PROVIDER: E-GEOD-13652 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.

Pan Qun Q Shai Ofer O Lee Leo J LJ Frey Brendan J BJ Blencowe Benjamin J BJ

Nature genetics 20081102 12

We carried out the first analysis of alternative splicing complexity in human tissues using mRNA-Seq data. New splice junctions were detected in approximately 20% of multiexon genes, many of which are tissue specific. By combining mRNA-Seq and EST-cDNA sequence data, we estimate that transcripts from approximately 95% of multiexon genes undergo alternative splicing and that there are approximately 100,000 intermediate- to high-abundance alternative splicing events in major human tissues. From a ...[more]

PMID: 18978789

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Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing

Project description:We carried out the first analysis of alternative splicing complexity in human tissues using mRNA-Seq data. New splice junctions were detected in 20% of multiexon genes, many of which are tissue specific. By combining mRNA-Seq and EST-cDNA sequence data, we estimate that transcripts from 95% of multiexon genes undergo alternative splicing and that there are 100,000 intermediate- to high-abundance alternative splicing events in major human tissues. From a comparison with quantitative alternative splicing microarray profiling data, we also show that mRNA-Seq data provide reliable measurements for exon inclusion levels. Keywords: mRNA expression

2008-11-19 | GSE13652 | GEO

Alternative Isoform Regulation in Human Tissue Transcriptomes

Project description:Through alternative processing of pre-mRNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes based on deep sequencing of cDNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analysis of mappings of sequence reads to exon-exon junctions indicated that ~94% of human genes undergo alternative splicing (AS), ~86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that a majority of AS and alternative cleavage and polyadenylation (APA) events exhibit variation between tissues. Variations in alternative mRNA isoform expression between 6 individuals were also detected in cerebellar cortex, with ~2- to 3-fold less isoform variation observed between individuals than between tissues. Extreme or 'switch-like' regulation of splicing between tissues was associated with increased sequence conservation and with generation of full-length open reading frames. Patterns of AS and APA were strongly correlated across tissues, suggesting coordinated regulation, and sequence conservation of known regulatory motifs in both regulated introns and 3' UTRs suggested common involvement of the same factors in regulation of tissue-specific splicing and polyadenylation. Exam mRNA expression in 15 human tissues and cell lines

2008-09-30 | E-GEOD-12946 | biostudies-arrayexpress

Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cells

Project description:Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, and that this has important functional and evolutionary implications. Activity of G-run SREs was higher for intermediate strength 5'ss by ~4-fold relative to weak 5'ss, and by ~1.3-fold relative to strong 5'ss. The dependence on 5'ss strength was supported both by comparative genomics and by microarray and Illumina mRNA-Seq analyses of splicing changes following RNAi against the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNP) H, which binds G-runs. This dependence implies that the responsiveness of exons to changes in hnRNP H levels is a bivariate function of both SRE abundance and 5'ss strength; this relationship may hold also for other splicing factors. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting both the frequency of 5'ss polymorphism and the evolution of new splicing patterns. Examine mRNA expression in 293T cells following hnRNP H or control siRNA knockdown

2009-09-12 | E-GEOD-16642 | biostudies-arrayexpress

Transcriptomic signatures of flight muscle, brain and leg tissues during Drosophila pupal development

Project description:Alternative splicing produces multiple, distinct mRNA transcripts from the same gene locus. To explore tissue-type specific gene expression and alternative splicing, we generated mRNA-Seq data from pupal tissues dissected from Drosophila. This allowed us to compare expression between dissected, highly enriched brain and indirect flight muscle (IFM) samples as well as whole legs. Bioinformatic analysis allowed us to characterize tissue-specific differences in gene expression as well as alternative splicing among the three tissues, as well as to identify a multitude of novel splicing events that are not currently contained in the Drosophila annotation. This suggests that tissue or cell-type specific sequencing at multiple timepoints during the fly life cycle will help extend existing annotations, and novel splice junction discovery is an important consideration in mRNA-Seq data analysis.

2022-01-25 | GSE194199 | GEO

Alternative expression analysis by RNA sequencing (comparison of 5-FU sensitive and resistant colorectal cancer cell lines)

Project description:In Alternative Expression Analysis (ALEXA)-Seq we developed a method to analyze massively parallel RNA sequence data to catalogue transcripts and assess differential and alternative expression of known and predicted messenger RNA (mRNA) isoforms in cells and tissues. As proof-of-principle, we applied the approach to a comparison of fluorouracil responsive and non-responsive human colorectal cancer cell lines. The sensitivity and specificity of the approach were assessed by comparison to exon tiling and splicing microarrays. Validations were conducted by reverse transcription polymerase chain reaction (PCR), quantitative PCR and Sanger sequencing. We observed global disruption of splicing in resistant cells characterized by expression of novel mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at our website: www.AlexaPlatform.org. See Griffith et al. 2010 in Nature Methods for further details.

2010-08-25 | GSE23776 | GEO

Alternative Isoform Regulation in Human Tissue Transcriptomes

2008-10-01 | GSE12946 | GEO

Identification of allele-specific alternative mRNA processing via transcriptome sequencing

Project description:Establishing the functional roles of genetic variants remains a significant challenge in the post-genomic era. Here, we present a method, allele-specific alternative mRNA processing (ASARP), to identify genetically influenced mRNA processing events using transcriptome sequencing (RNA-Seq) data. The method examines RNA-Seq data at both single nucleotide and whole-gene/isoform levels to identify allele-specific expression (ASE) and existence of allele-specific regulation of mRNA processing. We applied the methods to data obtained from the human glioblastoma cell line U87MG and primary breast cancer tissues and found that 26M-bM-^@M-^S45% of all genes with sufficient read coverage demonstrated ASE, with significant overlap between the two cell types. Our methods predicted potential mechanisms underlying ASE due to regulations affecting either whole-gene-level expression or alternative mRNA processing, including alternative splicing, alternative polyadenylation and alternative transcriptional initiation. Allele-specific alternative splicing and alternative polyadenylation may explain ASE in hundreds of genes in each cell type. Reporter studies following these predictions identified the causal single nucleotide variants (SNVs) for several allele-specific alternative splicing events. Finally, many genes identified in our study were also reported as disease/phenotype-associated genes in genome-wide association studies. Future applications of our approach may provide ample insights for a better understanding of the genetic basis of gene regulation underlying phenotypic diversity and disease mechanisms. Examine allele-specific gene expression and alternative RNA processing in U87MG cell line

2012-03-29 | E-GEOD-29738 | biostudies-arrayexpress

Project description:Alternative mRNA splicing

| PRJNA451020 | ENA

Widespread regulated alternative splicing of single codons accelerates proteome evolution

Project description:Thousands of human genes contain introns ending in NAGNAG motifs (N any nucleotide), where both NAGs can function as 3' splice sites, yielding isoforms differing by inclusion/exclusion of just three bases. However, the functional importance of NAGNAG alternative splicing is highly controversial. Using very deep RNA-Seq data from sixteen human and eight mouse tissues, we found that approximately half of alternatively spliced NAGNAGs undergo tissue-specific regulation and that regulated events have been selectively retained: alternative splicing of strongly tissue-specific NAGNAGs was ten times as likely to be conserved between species as for non-tissue-specific events. Further, alternative splicing of human NAGNAGs was associated with an order of magnitude increase in the frequency of exon length changes at orthologous mouse/rat exon boundaries, suggesting that NAGNAGs accelerate exon evolution. Together, our analyses show that NAGNAG alternative splicing constitutes a major generator of tissue-specific proteome diversity and accelerates evolution of proteins at exon-exon boundaries. mRNA-Seq of sixteen human and eight mouse tissues. Supplementary files: human.nagnag.junctions.gff and mouse.nagnag.junctions.gff are the annotation files (in GFF3 format) corresponding to the 'bwtout' mapped reads files linked to the Sample records. Raw data files provided for Samples GSM742937-GSM742952 only.

2012-01-05 | E-GEOD-30017 | biostudies-arrayexpress

Transcriptome sequencing reveals aberrant alternative splicing in Huntington's disease

Project description:Huntingtonâs disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component however has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of 7 human HD brains and 7 controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using an expanded panel of 54 brain tissues from patients and controls, we also identified 9 splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD RNA-seq analysis of the BA4 motor cortex of 7 control and 7 Huntington's disease patients. 1.5 ug of total RNA was used for RNA-seq library preparation using the TruSeqâ¢ Stranded mRNA LT Sample Prep Kit (Illumina). 100x2 bp paired-end RNA-seq reads were generated on a HiSeq 2000 sequencer.

2016-06-14 | E-GEOD-79666 | biostudies-arrayexpress

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