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 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 A0, Al, 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.
Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, Geneva, Switzerland. jesus.cruz@medecine.unige.ch
J
J
de la Cruz
D
D
Kressler
M
M
Rojo
D
D
Tollervey
P
P
Linder
D002466:Cell Nucleolus / Q000737:chemistry; D053487:DEAD-box RNA Helicases; D011132:Polyribosomes / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000096:biosynthesis; D012340:RNA, Ribosomal, 5.8S / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D012322:RNA Precursors; D012335:RNA, Ribosomal; D012340:RNA, Ribosomal, 5.8S; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C065039:RNA, ribosomal, 25S; D012316:RNA Nucleotidyltransferases; C063215:SPB4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases