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.