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

Hansen KD, Lareau LF, Blanchette M, Green RE, Meng Q, Rehwinkel J, Gallusser FL, Izaurralde E, Rio DC, Dudoit S, Brenner SE - PLoS Genet. (2009)

Bottom Line: Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biostatistics, School of Public Health, University of California Berkeley, Berkeley, CA, USA.

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.

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Isoform deconvolution.(A) Probe placement and gene structure for 3 NMD–affected genes: glorund, RpL10Ab, and squid. Gene structures are shown with exons as boxes and introns represented by peaked lines. Dark blue regions indicate the coding region and grey boxes show the untranslated regions (UTRs). Each probe is represented by a vertical colored line, and its complementary site on an isoform is shown by a half circle (exon probe) or full circle (splice junction probe). The different colors indicate the combination of isoforms each probe targets. The NMD–affected isoform of each gene is indicated. The coding sequences (CDSs) of CG6946-RC, CG7283-RB, and CG16901-RD were identified as described in the text. (B) Normalized log2 fold-changes for the probes in the upf1 experiment, grouped by which isoforms they target with colors corresponding to panel A. Each colored circle is the measurement of one probe on one array. The black circle is the group-wise mean of the fold-changes. (C) Deconvolved fold-change for the individual isoforms; “possibly absent” isoforms are not plotted (see text). (D) Estimated relative abundance of each present isoform in both the control and the NMD inhibited samples. We estimate that the NMD–target isoform of squid was a negligible fraction of total squid mRNA.
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pgen-1000525-g001: Isoform deconvolution.(A) Probe placement and gene structure for 3 NMD–affected genes: glorund, RpL10Ab, and squid. Gene structures are shown with exons as boxes and introns represented by peaked lines. Dark blue regions indicate the coding region and grey boxes show the untranslated regions (UTRs). Each probe is represented by a vertical colored line, and its complementary site on an isoform is shown by a half circle (exon probe) or full circle (splice junction probe). The different colors indicate the combination of isoforms each probe targets. The NMD–affected isoform of each gene is indicated. The coding sequences (CDSs) of CG6946-RC, CG7283-RB, and CG16901-RD were identified as described in the text. (B) Normalized log2 fold-changes for the probes in the upf1 experiment, grouped by which isoforms they target with colors corresponding to panel A. Each colored circle is the measurement of one probe on one array. The black circle is the group-wise mean of the fold-changes. (C) Deconvolved fold-change for the individual isoforms; “possibly absent” isoforms are not plotted (see text). (D) Estimated relative abundance of each present isoform in both the control and the NMD inhibited samples. We estimate that the NMD–target isoform of squid was a negligible fraction of total squid mRNA.

Mentions: Deconvolution requires probes targeting different combinations of isoforms. For a gene with only two isoforms, we require probes targeting the two individual isoforms as well as probes targeting both isoforms; having only probes targeting the individual isoforms would preclude the estimation of relative abundance. As an example of a situation where deconvolution is impossible, alternative polyadenylation can produce two isoforms that differ only in the length of the last exon, and there is no possible probe that uniquely targets the shorter isoform. For genes with more than two isoforms the details are more subtle, but in general a gene with isoforms requires probes targeting at least different combinations. This requirement makes it difficult to deconvolve genes with many isoforms, and, in some cases, it is provably impossible to obtain isoform-level fold-changes. Also, the algorithm and the array design assume that gene structures are known. Unknown alternative splice forms may lead to misinterpretation of the observed probe fold-changes. Examples of gene structures, probe locations, probe and isoform fold-changes, and relative proportions can be found in Figure 1.


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

Hansen KD, Lareau LF, Blanchette M, Green RE, Meng Q, Rehwinkel J, Gallusser FL, Izaurralde E, Rio DC, Dudoit S, Brenner SE - PLoS Genet. (2009)

Isoform deconvolution.(A) Probe placement and gene structure for 3 NMD–affected genes: glorund, RpL10Ab, and squid. Gene structures are shown with exons as boxes and introns represented by peaked lines. Dark blue regions indicate the coding region and grey boxes show the untranslated regions (UTRs). Each probe is represented by a vertical colored line, and its complementary site on an isoform is shown by a half circle (exon probe) or full circle (splice junction probe). The different colors indicate the combination of isoforms each probe targets. The NMD–affected isoform of each gene is indicated. The coding sequences (CDSs) of CG6946-RC, CG7283-RB, and CG16901-RD were identified as described in the text. (B) Normalized log2 fold-changes for the probes in the upf1 experiment, grouped by which isoforms they target with colors corresponding to panel A. Each colored circle is the measurement of one probe on one array. The black circle is the group-wise mean of the fold-changes. (C) Deconvolved fold-change for the individual isoforms; “possibly absent” isoforms are not plotted (see text). (D) Estimated relative abundance of each present isoform in both the control and the NMD inhibited samples. We estimate that the NMD–target isoform of squid was a negligible fraction of total squid mRNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2689934&req=5

pgen-1000525-g001: Isoform deconvolution.(A) Probe placement and gene structure for 3 NMD–affected genes: glorund, RpL10Ab, and squid. Gene structures are shown with exons as boxes and introns represented by peaked lines. Dark blue regions indicate the coding region and grey boxes show the untranslated regions (UTRs). Each probe is represented by a vertical colored line, and its complementary site on an isoform is shown by a half circle (exon probe) or full circle (splice junction probe). The different colors indicate the combination of isoforms each probe targets. The NMD–affected isoform of each gene is indicated. The coding sequences (CDSs) of CG6946-RC, CG7283-RB, and CG16901-RD were identified as described in the text. (B) Normalized log2 fold-changes for the probes in the upf1 experiment, grouped by which isoforms they target with colors corresponding to panel A. Each colored circle is the measurement of one probe on one array. The black circle is the group-wise mean of the fold-changes. (C) Deconvolved fold-change for the individual isoforms; “possibly absent” isoforms are not plotted (see text). (D) Estimated relative abundance of each present isoform in both the control and the NMD inhibited samples. We estimate that the NMD–target isoform of squid was a negligible fraction of total squid mRNA.
Mentions: Deconvolution requires probes targeting different combinations of isoforms. For a gene with only two isoforms, we require probes targeting the two individual isoforms as well as probes targeting both isoforms; having only probes targeting the individual isoforms would preclude the estimation of relative abundance. As an example of a situation where deconvolution is impossible, alternative polyadenylation can produce two isoforms that differ only in the length of the last exon, and there is no possible probe that uniquely targets the shorter isoform. For genes with more than two isoforms the details are more subtle, but in general a gene with isoforms requires probes targeting at least different combinations. This requirement makes it difficult to deconvolve genes with many isoforms, and, in some cases, it is provably impossible to obtain isoform-level fold-changes. Also, the algorithm and the array design assume that gene structures are known. Unknown alternative splice forms may lead to misinterpretation of the observed probe fold-changes. Examples of gene structures, probe locations, probe and isoform fold-changes, and relative proportions can be found in Figure 1.

Bottom Line: Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biostatistics, School of Public Health, University of California Berkeley, Berkeley, CA, USA.

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.

Show MeSH
Related in: MedlinePlus