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

被引:79
|
作者
de la Cruz, J
Kressler, D
Rojo, M
Tollervey, D
Linder, P
机构
[1] Ctr Med Univ Geneva, Dept Biochim Med, CH-1211 Geneva 4, Switzerland
[2] Univ Geneva, Dept Biochim, CH-1211 Geneva, Switzerland
[3] Univ Edinburgh, Inst Cell & Mol Biol, Edinburgh EH9 3JR, Midlothian, Scotland
关键词
DEAD-box proteins; pre-rRNA processing; ribosome biogenesis; yeast;
D O I
10.1017/S1355838298981158
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 underaccumulation 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 A(0), A(1), and A(2) 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.
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页码:1268 / 1281
页数:14
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