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Mol Cell Biol. 2015 Mar;35(5):816-30. doi: 10.1128/MCB.01348-14. Epub 2014 Dec 22.

DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

Author information

1
Département des sciences biologiques and Centre de recherche BioMed, Université du Québec à Montréal, Montreal, Quebec, Canada.
2
Department of Microbiology, Immunology and Cancer Biology, University of Virginia Health System, Charlottesville, Virginia, USA.
3
Département des sciences biologiques and Centre de recherche BioMed, Université du Québec à Montréal, Montreal, Quebec, Canada dragon.francois@uqam.ca.

Abstract

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

PMID:
25535329
PMCID:
PMC4323488
DOI:
10.1128/MCB.01348-14
[Indexed for MEDLINE]
Free PMC Article

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