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.In all organisms, the biogenesis of ribosomes is a fundamental process mediated by trans-acting factors. Experimental evidence strongly suggests that the ribosome synthesis process is well conserved throughout eukaryotes (reference 69 and references therein). Most of our knowledge on this process comes from studies performed with Saccharomyces cerevisiae (reviewed in references 16, 25, and 68). In this yeast, more than 225 protein trans-acting factors and 75 small nucleolar RNAs (snoRNAs) involved in ribosome synthesis have been identified (reviewed in references 16, 25, and 48). These factors, whose precise functions are still largely unknown, likely allow the ribosome maturation process to proceed with the required speed, accuracy and directionality (reviewed in references 30, 31, and 61).Biogenesis of eukaryotic ribosomes involves the transcription, processing, and modification of the precursors of rRNAs (pre-rRNAs), the folding and the assembly of the pre-rRNAs with the ribosomal proteins (r-proteins), and the export of the resulting preribosomal particles to the cytoplasm, where the last assembly reactions take place (16,25,39,44,63,65). Pre-rRNA processing is a well-defined pathway and involves a complex series of sequential endo-and exonucleolytic reactions (see Fig. S1 in the supplemental material). Much less is known about the reactions that allow formation, maturation, and movement of preribosomal particles through the nucleus and their subsequent export (see Fig. S2 in the supplemental material). However, the use of many different experimental approaches, in particular the purification of preribosomal particles ...

Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation

“…In clear contrast, L1 and L3 were present, as reported previously (61,90,116), in all tested preribosomal particles. Finally, and as expected, Nop1 is mainly associated with 90 S pre-ribosomal particles (114), Has1 with 90 S and early and intermediate pre-60 S r-particles (90), and Mrt4 with intermediate and late/cytoplasmic pre-60 S r-particles (61). Altogether, these results reveal that L40 associates with late/cytoplasmic pre-60 S r-particles but is only stably assembled into mature cytoplasmic 60 S r-subunits.…”

The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly

“…An equivalent mutation in the Spb4 helicase involved in yeast cytoplasmic ribosome assembly (Garcia-Gomez et al, 2011) or in the DbpA helicase required for bacterial ribosome assembly (Sharpe Elles et al, 2009) causes a dominant-negative phenotype when overexpressed in the corresponding wild-type strains. Overexpression of the DDX28 -R487A allele, but not of the wild-type allele, induced a mild but clear mt-LSU assembly defect, as seen by lowered steady-state levels of 16S rRNA and some mt-LSU mitoribosomal proteins (Fig 5E and F).…”

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…Most A3 and B factors assemble into early preribosomal particles containing 27SA 2 pre-rRNA (Saveanu et al 2003Zhang et al 2007;Garcia-Gomez et al 2011;Talkish et al 2012). Nsa2 and Nog2 assemble later, just before the ITS2 spacer is removed (Saveanu et al 2001;Lebreton et al 2006a).…”