Applications of High Throughput Nucleotide Sequencing

Background: Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. Results: We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMDdependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation. Conclusions: Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in coreand tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression. Background Alternative splicing (AS) involves the selective inclusion and exclusion of exons from a nascent pre-mRNA that results in various combinations of mature mRNAs with different coding potential and thus protein sequence [1]. Importantly, it has recently been estimated that nearly 95% of all multi-exon genes in the mammalian cell undergo AS [2,3], suggesting a pivotal role for AS in regulating and expanding the repertoire of isoforms expressed. By examining ESTs, it has been proposed that one-third of all AS isoforms contain a premature termination codon (PTC) [4], and these are expected to be targeted for degradation by nonsense-mediated mRNA decay (NMD). NMD is an mRNA quality control mechanism, and the primary function of NMD was initially thought to be in removal of aberrant transcripts arising from mutations or faulty transcription, mRNA processing or translation, but it is now evident that NMD impacts on both diverse physiological processes [5-7] as well as pathophysiological conditions (reviewed in [8]). The conserved core components of the NMD pathway are the UPF1, UPF2 and UPF3A/B proteins, and mutations or depletion of these factors inactivate NMD [9,10]. In mammalian cells, PTCs are distinguished from normal stop codons by their position relative to a downstream exon-exon junction, which is marked by the deposition of the exon junction complex * Correspondence: [email protected] † Contributed equally The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, DK2200 Copenhagen, Denmark Full list of author information is available at the end of the article Weischenfeldt et al. Genome Biology 2012, 13:R35 http://genomebiology.com/2012/13/5/R35 © 2012 Weischenfeldt et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.