SPB4P, AN ESSENTIAL PUTATIVE RNA HELICASE, IS REQUIRED FOR A LATE STEP IN THE ASSEMBLY OF 60S RIBOSOMAL-SUBUNITS IN SACCHAROMYCES-CEREVISIAE

Spb4p is a putative ATP-dependent RNA helicase that is required for sy nthesis of 60S ribosomal subunits. Polysome analyses of strains geneti cally 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, a nd primer extension indicated that these strains exhibited a reduced s ynthesis 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 tra nsfer 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 inh ibited. Sucrose gradient fractionation showed that the accumulated 27S B pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is l ocalized to the nucleolus and the adjacent nucleoplasmic area. On sucr ose gradients, HA-Spb4p was found almost exclusively in rapidly sedime nting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late an d essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.