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- Alalam, HannaGothenburg University,Göteborgs universitet,Institutionen för kemi och molekylärbiologi,Department of Chemistry and Molecular Biology (författare)
Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets.
- Artikel/kapitelEngelska2022
Förlag, utgivningsår, omfång ...
- 2022-03-16
- Cold Spring Harbor Laboratory,2022
Nummerbeteckningar
- LIBRIS-ID:oai:gup.ub.gu.se/314280
- https://gup.ub.gu.se/publication/314280URI
- https://doi.org/10.1261/rna.079077.121DOI
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- Språk:engelska
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- SwePubSwePub
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- Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
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- Zepeda-Martinez, Jorge Arturo (författare)
- Sunnerhagen, Per,1959Gothenburg University,Göteborgs universitet,Institutionen för kemi och molekylärbiologi,Department of Chemistry and Molecular Biology(Swepub:gu)xsuper (författare)
- Göteborgs universitetInstitutionen för kemi och molekylärbiologi (creator_code:org_t)
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- Ingår i:RNA: Cold Spring Harbor Laboratory28:71469-90011355-8382
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