Ofer Shai

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

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.

Deciphering the splicing code

Alternative splicing has a crucial role in the generation of biological complexity, and its misregulation is often involved in human disease. Here we describe the assembly of a ‘splicing code’, which uses combinations of hundreds of RNA features to predict tissue-dependent changes in alternative splicing for thousands of exons. The code determines new classes of splicing patterns, identifies distinct regulatory programs in different tissues, and identifies mutation-verified regulatory sequences. Widespread regulatory strategies are revealed, including the use of unexpectedly large combinations of features, the establishment of low exon inclusion levels that are overcome by features in specific tissues, the appearance of features deeper into introns than previously appreciated, and the modulation of splice variant levels by transcript structure characteristics. The code detected a class of exons whose inclusion silences expression in adult tissues by activating nonsense-mediated messenger RNA decay, but whose exclusion promotes expression during embryogenesis. The code facilitates the discovery and detailed characterization of regulated alternative splicing events on a genome-wide scale.

Functional coordination of alternative splicing in the mammalian central nervous system

BackgroundAlternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood.ResultsUsing quantitative AS microarray profiling, we have identified a large number of widely expressed mouse genes that contain single or coordinated pairs of alternative exons that are spliced in a tissue regulated fashion. The majority of these AS events display differential regulation in central nervous system (CNS) tissues. Approximately half of the corresponding genes have neural specific functions and operate in common processes and interconnected pathways. Differential regulation of AS in the CNS tissues correlates strongly with a set of mostly new motifs that are predominantly located in the intron and constitutive exon sequences neighboring CNS-regulated alternative exons. Different subsets of these motifs are correlated with either increased inclusion or increased exclusion of alternative exons in CNS tissues, relative to the other profiled tissues.ConclusionOur findings provide new evidence that specific cellular processes in the mammalian CNS are coordinated at the level of AS, and that a complex splicing code underlies CNS specific AS regulation. This code appears to comprise many new motifs, some of which are located in the constitutive exons neighboring regulated alternative exons. These data provide a basis for understanding the molecular mechanisms by which the tissue specific functions of widely expressed genes are coordinated at the level of AS.

Inferring global levels of alternative splicing isoforms using a generative model of microarray data

MOTIVATION Alternative splicing (AS) is a frequent step in metozoan gene expression whereby the exons of genes are spliced in different combinations to generate multiple isoforms of mature mRNA. AS functions to enrich an organisms proteomic complexity and regulates gene expression. Despite its importance, the mechanisms underlying AS and its regulation are not well understood, especially in the context of global gene expression patterns. We present here an algorithm referred to as the Generative model for the Alternative Splicing Array Platform (GenASAP) that can predict the levels of AS for thousands of exon skipping events using data generated from custom microarrays. GenASAP uses Bayesian learning in an unsupervised probability model to accurately predict AS levels from the microarray data. GenASAP is capable of learning the hybridization profiles of microarray data, while modeling noise processes and missing or aberrant data. GenASAP has been successfully applied to the global discovery and analysis of AS in mammalian cells and tissues. RESULTS GenASAP was applied to data obtained from a custom microarray designed for the monitoring of 3126 AS events in mouse cells and tissues. The microarray design included probes specific for exon body and junction sequences formed by the splicing of exons. Our results show that GenASAP provides accurate predictions for over one-third of the total events, as verified by independent RT-PCR assays. SUPPLEMENTARY INFORMATION http://www.psi.toronto.edu/GenASAP.

Abstract B67: Using alternative splicing microarrays to identify potential biomarkers in lung cancer

B67 The disruption of alternative splicing (AS) by either mutations in splicing sequences or changes in expression of splicing factors has been linked to many human diseases including cancer but the molecular changes associated with lung tumors are not well understood. Using our established quantitative AS microarray platform we have profiled matched normal and adenocarcinoma tissues from the lungs of 10 patients in order to identify changes at both the AS and transcriptional levels. These profiling experiments show a small set of exons that display pronounced and highly consistent changes between the normal and tumor tissues. This set is distinct from those genes showing changes at the transcriptional level. The results reveal how AS and transcription may be re-programmed during malignant transitions in the lung. Using a custom microarray with sets of exon body and splicing junction probes for profiling ~5000 human cassette alternative exons, we have identified 4 AS events that display pronounced inclusion level differences between normal and adenocarcinoma tissue in at least 80% of patients surveyed. These changes were confirmed by RT-PCR using the original 10 plus an additional 19 patient samples. Interestingly, all 4 of the alternative exons are located in genes that are linked to signaling pathways known to be deregulated in certain cancers. Moreover, these 4 AS events preserve frame and are conserved in mouse tissues, suggesting important functional roles. From the same dataset, a separate set of genes with transcript level changes were detected, consistent with previous findings that non-overlapping sets of genes are regulated at the AS and transcriptional levels, when comparing different tissues or corresponding tissues from different species. In addition to probe sets for profiling AS and transcript levels of the corresponding genes, our microarray contains probes to determine the transcript levels for 465 known and putative splicing factors. While significant changes in the expression levels of defined splicing factors were not detected, we observe changes in expression levels of 3 genes that contain RS domains, a feature of proteins that is predictive of a role in splicing. One of these genes is associated with the Notch pathway and the others are known tumor suppressor genes. We have confirmed that the change in AS for 1 of the target genes results in altered splicing at the protein level in addition to the RNA level changes. Work is in progress to characterize the functional role of this AS event using isoform-specific knockdown and overexpression. The increased expression of the exon-included protein isoform was evident in cell lines derived from both lung and colon cancer. We are currently determining if all 4 AS events occur in tumor tissue from breast and colon cancer patients. Also, we have expanded our profiling of human cancers by using higher density AS microarrays, and by using mRNA samples from additional normal and tumor-derived breast, colon and lung sources. Thus far, we have identified a set of conserved AS events that are consistently associated with lung adenocarcinomas. These are located in genes that function in signaling pathways that play important roles in tumorigenesis. Characterization of these AS events will advance our understanding of the role of splicing in human cancers and may also provide new targets for diagnostic applications as potential biomarkers. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B67.