Dynamics of the Putative RNA Helicase Spb4 during Ribosome Assembly in Saccharomyces cerevisiae

Abstract

Spb4 is a putative ATP-dependent RNA helicase that is required for proper processing of 27SB pre-rRNAs and therefore for 60S ribosomal subunit biogenesis. To define the timing of association of this protein with preribosomal particles, we have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, some protein factors of 90S preribosomal particles and the 35S and 27SA pre-rRNAs also copurify with TAP-tagged Spb4. Moreover, we have obtained by site-directed mutagenesis an allele that results in the R360A substitution in the conserved motif VI of the Spb4 helicase domain. This allele causes a dominant-negative phenotype when overexpressed in the wild-type strain. Cells expressing Spb4(R360A) display an accumulation of 35S and 27SB pre-rRNAs and a net 40S ribosomal subunit defect. TAP-tagged Spb4(R360A) displays a greater steady-state association with 90S preribosomal particles than TAP-tagged wild-type Spb4. Together, our data indicate that Spb4 is a component of early nucle(ol)ar pre-60S ribosomal particles containing 27SB pre-rRNA. Apparently, Spb4 binds 90S preribosomal particles and dissociates from pre-60S ribosomal particles after processing of 27SB pre-rRNA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.05436-11.

ACKNOWLEDGMENTS

We are indebted to all colleagues mentioned in the text for their gifts of material used in this study. We thank M. Dosil for advice on the affinity purification of GFP-tagged proteins and D. Kressler for critical reading of the manuscript.

This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN), FEDER (BFU2007-60151, BFU2010-15690, and HF2006-0153), and the Andalusian Government (CVI-271, P07-CVI-02623, and P08-CVI-03508) to J.D.L.C., from the CNRS, Université Paul Sabatier, the Agence Nationale de la Recherche, and the EGIDE Picasso Programme to Y.H., and from the Fondation pour la Recherche Médicale (Programme Grands Equipements) and the Génopole Toulose Midi-Pyrénées to B.M. J.J.G.-G. is a recipient of an FPI fellowship from MICINN.