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NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end non-templated nucleotides.
- Source :
-
Nature structural & molecular biology [Nat Struct Mol Biol] 2018 Oct; Vol. 25 (10), pp. 940-950. Date of Electronic Publication: 2018 Oct 01. - Publication Year :
- 2018
-
Abstract
- Nonsense-mediated messenger RNA decay (NMD) controls mRNA quality and degrades physiologic mRNAs to fine-tune gene expression in changing developmental or environmental milieus. NMD requires that its targets are removed from the translating pool of mRNAs. Since the decay steps of mammalian NMD remain unknown, we developed assays to isolate and sequence direct NMD decay intermediates transcriptome-wide based on their co-immunoprecipitation with phosphorylated UPF1, which is the active form of this essential NMD factor. We show that, unlike steady-state UPF1, phosphorylated UPF1 binds predominantly deadenylated mRNA decay intermediates and activates NMD cooperatively from 5'- and 3'-ends. We leverage method modifications to characterize the 3'-ends of NMD decay intermediates, show that they are ribosome-bound, and reveal that some are subject to the addition of non-templated nucleotide. Uridines are added by TUT4 and TUT7 terminal uridylyl transferases and removed by the Perlman syndrome-associated exonuclease DIS3L2. The addition of other non-templated nucleotides appears to inhibit decay.
- Subjects :
- Exoribonucleases genetics
Exoribonucleases metabolism
Exoribonucleases physiology
Exosome Multienzyme Ribonuclease Complex metabolism
Exosome Multienzyme Ribonuclease Complex physiology
HEK293 Cells
Humans
Models, Molecular
Nucleotidyltransferases metabolism
Nucleotidyltransferases physiology
Gene Expression Regulation
RNA Stability
RNA, Messenger metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1545-9985
- Volume :
- 25
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- Nature structural & molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 30275517
- Full Text :
- https://doi.org/10.1038/s41594-018-0132-7