Identification of Ribosome Biogenesis Factors Regulated by Nucleolar AAA-ATPase NVL2

Abstract

We previously reported that nucleolar AAA-ATPase NVL2 participates in 60S ribosome biogenesis and that its interaction with an RNA helicase MTR4/DOB1 may participate in this process. In general, AAA family ATPases act on a variety of intracellular complexes to facilitate their dissociation in an ATP dependent manner. Since MTR4/DOB1 is involved in the processing of rRNA precursors by cooperating with RNA exosome, a 3'-5' exonuclease complex, NVL2 may regulate these RNA-processing machineries. In this study, by using a proteomic analysis, we have screened for proteins, whose association with the complex are regulated by the NVL2 ATPase activity.First, HEK293 cells that can inducibly overexpress wild-type or mutated NVL2 were further transfected with an expression plasmid for FLAG-tagged MTR4/DOB1 to establish double-stable cell lines. Then MTR4/DOB1-associated proteins were purified from the cell extracts by coimmunoprecipitation using anti-FLAG immunoaffinity beads followed by MALDI-TOF MS protein identification. We were able to identify the known components of the exosome complex such as RRP6, RRP4, RRP40, RRP42, and RRP43. We next compared the protein components of these complexes among the cells expressing wild-type and mutated NVL2 by fluorescent two-dimensional differential gel electrophoresis (2D-DIGE) analysis. As a result, increased intensities of eleven protein spots were specifically detected by expressing an ATP hydrolysis mutant of NVL2 (E365Q/E682Q). These proteins might be potential targets of NVL2, which are dissociated from the MTR4/DOB1-containing complexes during ribosome biogenesis in conjunction with NVL2 ATP hydrolysis.