RNA



Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae


JESÚS DE LA  CRUZ a1 1 c1, DIETER  KRESSLER a1 1 , MANUEL  ROJO a2, DAVID  TOLLERVEY a3 and PATRICK  LINDER a1
a1 Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, CH-1211 Geneva, Switzerland
a2 Département de Biochimie, Sciences II, Université de Genève, CH-1211 Geneva, Switzerland
a3 Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom

Abstract

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an under-accumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, A1, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.

(Accepted July 13 1998)


Key Words: DEAD-box proteins; pre-rRNA processing; ribosome biogenesis; yeast.

Correspondence:
c1 Reprint requests to: Jesús de la Cruz, Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève; 1, rue Michel Servet, CH-1211 Geneva 4, Switzerland; e-mail: jesus.cruz@medecine.unige.ch; web page address: http://www.expasy.ch/linder/.


Footnotes

1 The first two authors contributed equally to the work.