Unknown,Transcriptomics,Genomics,Proteomics

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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 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Eric Wang

PROVIDER: E-GEOD-23694 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

Katz Yarden Y Wang Eric T ET Airoldi Edoardo M EM Burge Christopher B CB

Nature methods 20101107 12

Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splici ...[more]

PMID: 21057496

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Project description:Autism spectrum disorder (ASD) is a common, highly heritable neurodevelopmental condition characterized by marked genetic heterogeneity. Thus, a fundamental question is whether autism represents an aetiologically heterogeneous disorder in which the myriad genetic or environmental risk factors perturb common underlying molecular pathways in the brain. Here, we demonstrate consistent differences in transcriptome organization between autistic and normal brain by gene co-expression network analysis. Remarkably, regional patterns of gene expression that typically distinguish frontal and temporal cortex are significantly attenuated in the ASD brain, suggesting abnormalities in cortical patterning. We further identify discrete modules of co-expressed genes associated with autism: a neuronal module enriched for known autism susceptibility genes, including the neuronal specific splicing factor A2BP1 (also known as FOX1), and a module enriched for immune genes and glial markers. Using high-throughput RNA sequencing we demonstrate dysregulated splicing of A2BP1-dependent alternative exons in the ASD brain. Moreover, using a published autism genome-wide association study (GWAS) data set, we show that the neuronal module is enriched for genetically associated variants, providing independent support for the causal involvement of these genes in autism. In contrast, the immune-glial module showed no enrichment for autism GWAS signals, indicating a non-genetic aetiology for this process. Collectively, our results provide strong evidence for convergent molecular abnormalities in ASD, and implicate transcriptional and splicing dysregulation as underlying mechanisms of neuronal dysfunction in this disorder. To identify potential A2BP1-dependent differential splicing events in ASD brain, we performed high-throughput RNA sequencing (RNA-Seq) on three autism samples with significant downregulation of A2BP1 (average fold change by quantitative RT-PCR = 5.9) and three control samples with average A2BP1 levels. The list of potential A2BP1-depending differential splicing events in ASD is given in the Supplementary file linked at the foot of this record.

Dataset Information

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

Publications

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

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