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ABSTRACT: Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.
Nucleic Acids Research 12/2012; · 8.03 Impact Factor
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ABSTRACT: Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.
Molecular and cellular biology 06/2012; 32(16):3228-41. · 6.06 Impact Factor
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ABSTRACT: Gene expression in eukaryotes is an essential process that includes transcription, RNA processing, and export. One important player in this interface is the poly(A)(+)-RNA-binding protein Nab2, which regulates the mRNA poly(A)(+)-tail length and export. Here we show that Nab2 has additional roles during mRNA transcription, tRNA metabolism, and ribosomal subunit export. Nab2 is associated with the entire open reading frame of actively transcribed RNA polymerase (RNAP) II and III genes. As a consequence, nab2 mutations confer translation defects that are detected by polysome profiling. Genome-wide analysis of expression of a conditional degron nab2 mutant shows that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is direct. Taken together, our results identify novel functions for Nab2 in transcription and metabolism of most types of RNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, and that it is a central player in the general and coordinated control of gene expression from transcription to translation.
Molecular biology of the cell 06/2011; 22(15):2729-40. · 5.98 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae, ribosome biogenesis requires, in addition to rRNA and ribosomal proteins, a myriad of small nucleolar RNAs (snoRNAs) and over two hundred protein trans-acting factors. There are protein trans-acting factors predicted to participate in ribosome biogenesis that have not been so far characterized. Here, we report the functional analysis of the Nucleolar protein 6 (Nop6) in ribosome biogenesis. Our results show that Nop6 is needed for optimal 40S ribosomal subunit biogenesis. Deletion of NOP6 leads to an appropriate 20% reduction in 18S rRNA levels and therefore in 40S ribosomal subunits. This is due to mild inhibition of pre-rRNA processing at cleavage site A 2. Tandem affinity purification followed by mass spectrometry and northern blot analyses indicate that Nop6 is a component of 90S pre-ribosomal particles. rDNA chromatin immunoprecipitation experiments and analysis of the intracellular localisation of Nop6-eGFP after in vivo shut down of pre-rRNA transcription strongly suggest that Nop6 binds to the pre-rRNA early during transcription. Genetic data suggest that Nop6 and the snoRNA snR57 both interact similarly with the protein trans-acting factor Nep1. It has been proposed that snR57 and Nep1 participate in a pre-rRNA conformational switch that allows the proper assembly of 40S ribosomal protein S19. Our results strongly suggest that the role Nop6 might have in this conformational switch is independent of snR57.
RNA biology 01/2011; 8(1):112-24. · 5.56 Impact Factor
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ABSTRACT: Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C(2).
Nucleic Acids Research 08/2010; 38(15):5177-92. · 8.03 Impact Factor
