Regulated endocleavage balances the degradation of nonsense-mediated decay substrates
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Homo sapiens
SUBMITTER: Jernej Ule
PROVIDER: E-MTAB-3625 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We aimed to study the cleavage sites of nonsense-mediated mRNA decay (NMD) substrates. Therefore, we depleted the major exoribonuclease XRN1 in human cell culture, which degrades the 3' fragments generated by SMG6-mediated endonucleolytic cleavage. Different reporter mRNAs or endogenous NMD targets were investigated and the 3' fragments were cloned, amplified and subsequently subjected to high throughput sequencing.
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Project description:Mature mRNAs undergo quality control during translation that may lead to RNA degradation by triggering the nonsense mediated decay (NMD) pathway. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction or an intron in the 3'UTR activates NMD. However, many of the features that lead to the activation of this pathway are unclear. UPF1, an RNA helicase, is the core NMD factor. UPF1 forms a multi-protein complex by recruiting a series of factors and other protein complexes in a process that depends on the UPF1 phosphorylation-dephosphorylation cycle. Among the factors recruited by UPF1, SMG5-SMG7 and SMG6 have critical importance in executing NMD. The SMG5-SMG7 heterodimer induces the exonucleolytic degradation of the mRNA, which depends on the recruitment of deadenylation factors. SMG6 has endonucleolytic activity and cleaves the targeted transcript close to the stop codon. The redundancy between the exonucleolytic and endonucleolytic paths to achieve degradation during NMD has been previously reported in the literature. To investigate the apparent redundancy between SMG5-SMG7 and SMG6 activity and to further understand the features that lead to the activation of NMD, we have generated two clones of SMG7 knockout human cells using CRISPR-Cas9. We generated mRNA-Sequencing data for control and both SMG7 KO clones with additional siRNA-mediated knockdown of Luciferase (Luc) as control, SMG5 or SMG6.
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Project description:Nonsense-mediated RNA decay (NMD) is a conserved RNA turnover pathway. Here we report that the sole endonuclease in the NMD pathway, SMG6, is essential for the male germline. Germ-cell conditional knockout (cKO) of Smg6 causes complete arrest of spermatogenesis at the early haploid cell stage. Smg6-cKO round spermatids accumulate NMD target mRNAs with long 3’ untranslated regions (UTRs) and fail to eliminate transcripts normally expressed during meiosis, the previous step in spermatogenesis. SMG6 and PIWI-interacting (pi) RNA pathway components are highly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells. This led to the intriguing possibility that the CB is a site where SMG6 and the piRNA pathway co-operate to regulate RNA metabolism. Several findings supported this hypothesis: (i) SMG6 and the piRNA-binding protein PIWIL1 have almost identical temporal expression and localization patterns, (ii) SMG6 and PIWIL1 physically interact, (iii) scores of genes upregulated in Smg6-cKO round spermatids overlap with those upregulated in Piwil1-KO round spermatids, and (iv) the phenotypic defects caused by SMG6 loss phenocopies the defects caused by PIWIL1 loss. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.
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