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.
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.
Schell
Thomas
T
European Molecular Biology Laboratory Heidelberg, Gene Expression Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Köcher
Thomas
T
Wilm
Matthias
M
Seraphin
Bertrand
B
Kulozik
Andreas E
AE
Hentze
Matthias W
MW
D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D020365:RNA Helicases; D012333:RNA, Messenger / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D020365:RNA Helicases; C101828:UPF1 protein, human