The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis (original) (raw)
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The EMBO Journal, 2011
The precise functions of most of the B200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assemblyprocessing of 27SA 3 pre-rRNA. Six of seven assembly factors required for this step (A 3 factors) are mutually interdependent for association with preribosomes. These A 3 factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A 3 factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A 3 factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs.
Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits
Nucleic acids research, 2014
During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assembl...
A Pre-Ribosome with a Tadpole-like Structure Functions in ATP-Dependent Maturation of 60S Subunits
Molecular Cell, 2004
small and large subunits' pathways diverge (Venema and Tollervey, 1999). Both pre-60S biogenesis and pre-40S biogenesis have then been followed by biochemical purification of intermediate particles (Baßler et al.Im Neuenheimer Feld 328 69120 Heidelberg Germany et al., 2002; Nissan et al., 2002; Schä fer et al., 2002). A large number of non-ribosomal proteins that have been 2 Biozentrum M.E. Mü ller Institute for Structural Biology identified in pre-60S particles have no assigned function in RNA metabolism. However, many of these factors CH-4056 Basel Switzerland have intriguing motifs, such as putative GTPases and AAA-type ATPases (Tschochner and Hurt, 2003). The 3 Wellcome Trust Centre for Cell Biology University of Edinburgh number of non-ribosomal proteins associated with pre-60S particles decreases during the maturation process, EH9 3JR Edinburgh Scotland from more than 30 components initially present on early particles to a handful of proteins associated with late particles that are close to nuclear export. The rix1-1 mutation was identified in a screen for tem-Summary perature-sensitive (ts) strains with defects in nuclear export of 60S subunits (Baßler et al., 2001; Gadal et Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S
Journal of Biological Chemistry, 2012
Background: The contribution of ribosomal proteins to ribosome assembly and function is often not well understood. Results: L40 assembles within the cytoplasm into pre-60 S subunits and is required for Nmd3 and Rlp24 recycling. Conclusion: L40 contributes to formation of 60 S subunits competent for subunit joining and translation elongation. Significance: Our analysis of L40 function reveals an additional step during cytoplasmic pre-60 S maturation events.
Journal of Biological Chemistry, 2008
The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and 32 Plabeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells.
Molecular and cellular biology, 1993
Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another...
Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation
RNA, 2004
The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA 3 to 27SB S pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA 3 and 27SB L pre-ribosomal RNAs.
RNA (New York, N.Y.), 2016
In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of nascent pre-rRNAs are processed cotranscriptionally, with cleavage at the A2site first releasing a pre-40S ribosomal subunit followed by release of a pre-60S ribosomal subunit upon transcription termination. Ribosome assembly is driven in part by hierarchical association of assembly factors and r-proteins. Groups of proteins are thought to associate with pre-ribosomes cotranscriptionally during early assembly steps, whereas others associate later, after transcription is completed. Here we describe a previously uncharacterized phenotype observed upon disruption of ribosome assembly, in which normally late-binding proteins associate earlier, with pre-ribosomes containing 35S pre-rRNA. As previously observed by many other groups, we show that disruption of 60S subunit biogenesis results in increased amounts of 35S pre-rRNA, sugge...
2000
The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino ac- ids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly ex- pressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribo- somal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is re- quired for proper processing of pre-rRNA and the assembly of preribosomal 40S sub- units. In the course of functional analysis of un- known open reading frames found during the sequencing of chromosome III of Saccharo- myces cerevisiae we have identified a novel gene KRR1 (YCL059c) required for cell viabil- ity. It encodes a protein containing a charac- teristic nuclear targeting sequence highly con- served...
Molecular Microbiology, 2004
SummaryThe Has1 protein, a member of the DEAD‐box family of ATP‐dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre‐60S ribosomal complexes. Here, we show that Has1p is an essential trans‐acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature‐sensitive has1‐1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre‐rRNA processing by pulse‐chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre‐rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre‐rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a...