DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

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 the U14 small nucleolar (sno) RNA to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole cell extracts (WCEs) revealed that 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 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 co-transcriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences on 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.