RNA



Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure


NICHOLAS J.  WATKINS a1, ALEXANDER  GOTTSCHALK a1, GITTE  NEUBAUER a2, BERTHOLD  KASTNER a1, PATRIZIA  FABRIZIO a1, MATTHIAS  MANN a2 and REINHARD  LÜHRMANN a1c1
a1 Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil Mannkopff-Straße 2, D35037 Marburg, Germany
a2 European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, 69012 Heidelberg, Germany

Abstract

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in pre-ribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine ([Psi]) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic [Psi] synthases. The presence of Cbf5p, a putative [Psi] synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.

(Received May 12 1998)
(Revised June 29 1998)
(Accepted September 11 1998)


Key Words: ribosomal RNA processing; RNA-binding proteins; rRNA modification; Saccharomyces cerevisiae; small nucleolar RNAs.

Correspondence:
c1 Reprint requests to: Reinhard Lührmann, Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil Mannkopff-Straße 2, D35037 Marburg, Germany; e-mail: luehrmann@imt.uni-marburg.de.