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The Substrates of Nonsense-Mediated mRNA Decay in Caenorhabditis elegans.

ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a conserved pathway that strongly influences eukaryotic gene expression. Inactivating or inhibiting NMD affects the abundance of a substantial fraction of the transcriptome in numerous species. Transcripts whose abundance is altered in NMD-deficient cells may represent either direct substrates of NMD or indirect effects of inhibiting NMD. We present a genome-wide investigation of the direct substrates of NMD in Caenorhabditis elegans Our goals were (i) to identify mRNA substrates of NMD and (ii) to distinguish those mRNAs from others whose abundance is indirectly influenced by the absence of NMD. We previously demonstrated that Upf1p/SMG-2, the central effector of NMD in all studied eukaryotes, preferentially associates with mRNAs that contain premature translation termination codons. We used this preferential association to distinguish direct from indirect effects by coupling immunopurification of Upf1/SMG-2 with high-throughput mRNA sequencing of NMD-deficient mutants and NMD-proficient controls. We identify 680 substrates of NMD, 171 of which contain novel spliced forms that (i) include sequences of annotated introns and (ii) have not been previously documented in the C. elegans transcriptome. NMD degrades unproductively spliced mRNAs with sufficient efficiency in NMD-proficient strains that such mRNAs were not previously known. Two classes of genes are enriched among the identified NMD substrates: (i) mRNAs of expressed pseudogenes and (ii) mRNAs of gene families whose gene number has recently expanded in the C. elegans genome. Our results identify novel NMD substrates and provide a context for understanding NMD's role in normal gene expression and genome evolution.

SUBMITTER: Muir VS

PROVIDER: S-EPMC5765348 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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The Substrates of Nonsense-Mediated mRNA Decay in <i>Caenorhabditis elegans</i>.

Muir Virginia S VS Gasch Audrey P AP Anderson Philip P

G3 (Bethesda, Md.) 20180104 1

Nonsense-mediated mRNA decay (NMD) is a conserved pathway that strongly influences eukaryotic gene expression. Inactivating or inhibiting NMD affects the abundance of a substantial fraction of the transcriptome in numerous species. Transcripts whose abundance is altered in NMD-deficient cells may represent either direct substrates of NMD or indirect effects of inhibiting NMD. We present a genome-wide investigation of the direct substrates of NMD in <i>Caenorhabditis elegans</i> Our goals were (i ...[more]

PMID: 29122854

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Project description:Purpose: We present a genome-wide investigation to distinguish mRNA substrates directly regulated by nonsense-mediated mRNA decay (NMD) from indirect secondary effects using true NMD null alleles Methods: mRNA profiles of wild type (N2), smg-1(r910), and smg-1(r910) smg-2(r915) animals were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. We further performed deep sequencing in triplicate on RNAs that co-immunoprecipitate with the central effector of NMD, SMG-2, in both smg-1(r910) and smg-1(r910) smg-2(r915) mutant animals. The sequence reads that passed quality filters were analyzed at both the gene level and the intron level using Tophat2 and edgeR. qRT-PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: We found that a significant portion of genes with increased expression in an NMD-deficient background are direct substrates of SMG-2. Among the list of direct targets, we find many mRNAs from pseudogenes, alternative splice isoforms harboring PTCs, mRNAs encoding RNA processing factors, and two recently expanded gene families are directly regulated by the NMD pathway.

Dataset Information

The Substrates of Nonsense-Mediated mRNA Decay in Caenorhabditis elegans.

Publications

The Substrates of Nonsense-Mediated mRNA Decay in <i>Caenorhabditis elegans</i>.

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