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PMID: 12723973 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells.

The Biochemical journal ·Vol. 373 ·No. Pt 3 ·2003-08-01 ·Pages 775-83

Schell T, Köcher T, Wilm M, Seraphin B, Kulozik AE, Hentze MW

Abstract

mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag). Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent. Using affinity chromatography, Hupf1-2tag-associated proteins were isolated. MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1. Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx. 1.3 MDa. Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag. In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase. Components of the exon-exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag. We discuss these findings in the context of current models of NMD.

MeSH Terms
Base Sequence Chromatography, Gel DNA Primers Electrophoresis, Polyacrylamide Gel Fluorescent Antibody Technique HeLa Cells Humans Mass Spectrometry RNA Helicases RNA, Messenger/metabolism Trans-Activators Transcription Factors/isolation & purification,metabolism
Chemicals
DNA Primers RNA, Messenger Trans-Activators Transcription Factors RNA Helicases UPF1 protein, human
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Schell Thomas
European Molecular Biology Laboratory Heidelberg, Gene Expression Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Köcher Thomas
Wilm Matthias
Seraphin Bertrand
Kulozik Andreas E
Hentze Matthias W
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/BJ20021920
Published
2003-08-01
Pages
775-83
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1223536
Subset
IM
Corrections
CommentIn
PMID: 12939168
External Links
PubMed source DOI: 10.1042/BJ20021920 PMC Full Text View journal details
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