Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in Drosophila

Kasper Daniel Hansen,Elisa Izaurralde,Liana F Lareau,Sandrine Dudoit,Jan Rehwinkel,Qi Meng,Fabian L Gallusser,Richard E Green,Donald C Rio,Marco Blanchette,Steven E Brenner

doi:10.1371/journal.pgen.1000525

Abstract

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly.

Highlights

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance system that down-regulates mRNAs containing early stop codons in all eukaryotes examined [1]
In order to identify cellular mRNAs naturally targeted by the nonsense-mediated mRNA decay (NMD) machinery, RNA was obtained from a previous experiment in which levels of the key NMD effectors UPF1 and UPF2 were reduced in S2 cells by dsRNAi, with three independent knockdowns of each effector [20]
Following the functional knockdown of the NMD machinery, confirmed by the stabilization of an NMD reporter, RNA was extracted and the microarray was used to probe the changes in alternative splicing patterns relative to the patterns in control cells treated with an unrelated dsRNA

Summary

Introduction

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance system that down-regulates mRNAs containing early stop codons in all eukaryotes examined [1]. NMD functions to clear the cell of transcripts containing potentially harmful nonsense mutations [2] In addition to this role in surveillance of mutations, NMD affects the expression of numerous non-mutant endogenous targets [3,4,5,6]. These natural targets include many mRNAs that are the products of alternative splicing; one study reported that 45% of alternatively spliced human genes have at least one isoform that may be degraded by NMD [7]. Unproductive splicing is used in the regulation and autoregulation of numerous genes [8] including mammalian splicing factors, spliceosome components [9,10,11,12,13,14] and the spermidine/spermine N1-acetyltransferase (SSAT) gene [15]

Methods

Results

Conclusion