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Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates.

ABSTRACT: Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3' fragment capture and degradome sequencing. This reveals that endogenous transcripts can have NMD-eliciting features at various positions, including upstream open reading frames (uORFs), premature termination codons (PTCs), and long 3' UTRs. We find that NMD substrates with PTCs undergo constitutive SMG6-dependent endocleavage, rather than SMG7-dependent exonucleolytic decay. In contrast, the turnover of NMD substrates containing uORFs and long 3' UTRs involves both SMG6- and SMG7-dependent endo- and exonucleolytic decay, respectively. This suggests that the extent to which SMG6 and SMG7 degrade NMD substrates is determined by the mRNA architecture.

SUBMITTER: Ottens F

PROVIDER: S-EPMC5513067 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates.

Ottens Franziska F Boehm Volker V Sibley Christopher R CR Ule Jernej J Gehring Niels H NH

RNA (New York, N.Y.) 20170501 8

Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via ...[more]

PMID: 28461625

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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.

Project description:Proinflammatory cytokines are implicated in pancreatic β-cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of Nonsense-Mediated RNA Decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in β-cells. A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-βH3 or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. Gain- or loss-of function of two key NMD components UPF3B and UPF2 is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Cyt attenuate NMD activity in insulin-producing cell lines and primary human β-cells. These effects are found to involve ER stress and are associated with downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing, raises or lowers Cyt-induced cell death, respectively, in EndoC-βH3 cells, and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increase alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in extracellular matrix (ECM) including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitises β-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signalling, potentially serving as a protective response against Cyt-induced NMD component expression. Our findings highlight the central importance of RNA turnover in β-cell responses to inflammatory stress.

Dataset Information

Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates.

Publications

Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates.

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