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A Role for the Nonsense-Mediated mRNA Decay Pathway in Maintaining Genome Stability in Caenorhabditis elegans.

ABSTRACT: Ionizing radiation (IR) is commonly used in cancer therapy and is a main source of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA damage. We have used Caenorhabditis elegans as an invertebrate model to identify novel factors required for repair of DNA damage inflicted by IR. We have performed an unbiased genetic screen, finding that smg-1 mutations confer strong hyper-sensitivity to IR. SMG-1 is a phosphoinositide-3 kinase (PI3K) involved in mediating nonsense-mediated mRNA decay (NMD) of transcripts containing premature stop codons and related to the ATM and ATR kinases which are at the apex of DNA damage signaling pathways. Hyper-sensitivity to IR also occurs when other genes mediating NMD are mutated. The hyper-sensitivity to bleomycin, a drug known to induce DSBs, further supports that NMD pathway mutants are defective in DSB repair. Hyper-sensitivity was not observed upon treatment with alkylating agents or UV irradiation. We show that SMG-1 mainly acts in mitotically dividing germ cells, and during late embryonic and larval development. Based on epistasis experiments, SMG-1 does not appear to act in any of the three major pathways known to mend DNA DSBs, namely homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ). We speculate that SMG-1 kinase activity could be activated following DNA damage to phosphorylate specific DNA repair proteins and/or that NMD inactivation may lead to aberrant mRNAs leading to synthesis of malfunctioning DNA repair proteins.

SUBMITTER: Gonzalez-Huici V

PROVIDER: S-EPMC5560793 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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A Role for the Nonsense-Mediated mRNA Decay Pathway in Maintaining Genome Stability in Caenorhabditis elegans.

González-Huici Víctor V Wang Bin B Gartner Anton A

Genetics 20170620 4

Ionizing radiation (IR) is commonly used in cancer therapy and is a main source of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA damage. We have used Caenorhabditis elegans as an invertebrate model to identify novel factors required for repair of DNA damage inflicted by IR. We have performed an unbiased genetic screen, finding that smg-1 mutations confer strong hyper-sensitivity to IR. SMG-1 is a phosphoinositide-3 kinase (PI3K) involved in mediating nonsense- ...[more]

PMID: 28634159

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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

A Role for the Nonsense-Mediated mRNA Decay Pathway in Maintaining Genome Stability in Caenorhabditis elegans.

Publications

A Role for the Nonsense-Mediated mRNA Decay Pathway in Maintaining Genome Stability in <i>Caenorhabditis elegans</i>.

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