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Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.

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.

SUBMITTER: Hansen KD

PROVIDER: S-EPMC2689934 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.

Hansen Kasper Daniel KD Lareau Liana F LF Blanchette Marco M Green Richard E RE Meng Qi Q Rehwinkel Jan J Gallusser Fabian L FL Izaurralde Elisa E Rio Donald C DC Dudoit Sandrine S Brenner Steven E SE

PLoS genetics 20090619 6

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 ...[more]

PMID: 19543372

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Project description:Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3' untranslated region (UTR), along with exon seven. Therefore, this 3' UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3'-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites--two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.

Project description:A major task in analyzing alternative splicing (AS) by RNA-Seq is to explore and quantify the differential representation of various splice classes and the protein-coding potential of the mapped reads. To this end, we have generated a streamlined bioinformatic pipeline available to the scientific community, which maps RNA-Seq reads to a complex combinatorial database of exon-exon junctions for the unique junction discovery, PTC detection and identification of splice isoforms. Hence, we have incorporated a splice isoform inference and PTC detection algorithm, which facilitates a highly accurate mapping and prediction of splice isoform junctions and nonsense-mediated mRNA decay (NMD)-susceptibility. We used this pipeline to investigate the complexity of the transcriptome and global role of AS-NMD in vivo in mammalian cells, by taking advantage of our Upf2 conditional knock-out mouse. Using tissue-specific Cre deleter strains, we have previously demonstrated in vivo that NMD acts to degrade many transcripts that results from AS. Also, ablating UPF2 (a core NMD factor) in the mouse leads to rapid mortality and collapse of most organs tested (Thoren et al., 2010; Weischenfeldt et al., 2008), suggesting an essential function for NMD in AS. To explore the effect of NMD on global splicing and to generate and validate an attractive bioinformatic pipeline for the scientific community to study AS and NMD, we chose to analyze two different mammalian organ systems with distinct phenotypes upon UPF2 ablation to test the robustness and dynamic range of the analysis. In one end of the spectrum, we chose to analyze the liver, wherein removal of UPF2 results in failure in liver metabolism and a high mortality rate (Thoren et al., 2010). To profile a less affected tissue, we chose to analyze bone marrow-derived macrophages (BMMs). These are macrophages that differentiate in vitro and Upf2 deleted BMMs are completely devoid of NMD activity but nevertheless show no morphological or functional phenotype compared to wild-type controls (Weischenfeldt et al., 2008). Thus, we were able to make a direct comparison between these two tissues to demonstrate the potency of our Isoform and PTC detection pipeline described below, which will allow the scientific community to analyze transcriptome data for global AS and NMD-susceptibility. Examination of UPF2 WT vs. KO in two different murine tissues (liver, bone marrow-derived macrophages).

Dataset Information

Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.

Publications

Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.

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