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

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.
Mesh Terms:
Centrifugation, Density Gradient, Chromatin, DEAD-box RNA Helicases, Genotype, Microscopy, Electron, Phosphoproteins, Protein Structure, Tertiary, RNA Helicases, RNA Nucleotidyltransferases, RNA, Ribosomal, 18S, RNA, Small Nucleolar, Ribonucleoproteins, Ribosomes, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Mol. Cell. Biol.
Date: Mar. 01, 2015
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