Yeast Pescadillo is required for multiple activities during 60S ribosomal subunit synthesis (original) (raw)
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Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation
RNA, 2005
In eukaryotes ribosome biogenesis required that rRNAs primary transcripts are assembled in pre-ribosomal particles and processed. Protein factors and pre-ribosomal complexes involved in this complex pathway are not completely depicted. The essential ORF YPR143W encodes in yeast for an uncharacterized protein product, named here Rrp15p. Cellular function of Rrp15p has not so far defined even if nucleolar location was referred. With the aim to define the possible role of this orphan gene, we performed TAP-tagging of Rrp15p and investigated its molecular association with known pre-ribosomal complexes. Comparative sucrose gradient sedimentation analyses of yeast lysates expressing the TAP-tagged Rrp15p, strongly indicated that this protein is a component of the pre-60S particles. Northern hybridization, primer extension and functional proteomics on TAP-affinity isolated complexes proved that Rrp15p predominately associated with pre-rRNAs and proteins previously characterized as componen...
Nop53p is a novel nucleolar 60S ribosomal subunit biogenesis protein
Biochemical Journal, 2005
Ribosome biogenesis in Saccharomyces cerevisiae occurs primarily in a specialized nuclear compartment termed the nucleolus within which the rRNA genes are transcribed by RNA polymerase I into a large 35 S rRNA precursor. The ensuing association/dissociation and catalytic activity of numerous trans-acting protein factors, RNAs and ribosomal proteins ultimately leads to the maturation of the precursor rRNAs into 25, 5.8 and 18 S rRNAs and the formation of mature cytoplasmic 40 and 60 S ribosomal subunits. Although many components involved in ribosome biogenesis have been identified, our understanding of this essential cellular process remains limited. In the present study we demonstrate a crucial role for the previously uncharacterized nucleolar protein Nop53p (Ypl146p) in ribosome biogenesis. Speci-fically, Nop53p appears to be most important for biogenesis of the 60 S subunit. It physically interacts with rRNA processing factors, notably Cbf5p and Nop2p, and co-fractionates specifically with pre-60 S particles on sucrose gradients. Deletion or mutations within NOP53 cause significant growth defects and display significant 60 S subunit deficiencies, an imbalance in the 40 S:60 S ratio, as revealed by polysome profiling, and defects in progression beyond the 27 S stage of 25 S rRNA maturation during 60 S biogenesis.
Proceedings of the National Academy of Sciences, 2000
The Saccharomyces cerevisiae Rlp7 protein has extensive identity and similarity to the large ribosomal subunit L7 proteins and shares an RNA-binding domain with them. Rlp7p is not a ribosomal protein; however, it is encoded by an essential gene and therefore must perform a function essential for cell growth. In this report, we show that Rlp7p is a nucleolar protein that plays a critical role in processing of precursors to the large ribosomal subunit RNAs. Pulse–chase labeling experiments with Rlp7p-depleted cells reveal that neither 5.8S S , 5.8S L , nor 25S is produced, indicating that both the major and minor processing pathways are affected. Analysis of processing intermediates by primer extension indicates that Rlp7p-depleted cells accumulate the 27SA 3 precursor RNA, which is normally the major substrate (85%) used to produce the 5.8S and 25S rRNAs, and the ratio of 27SB L to 27SB S precursors changes from approximately 1:8 to 8:1 (depleted cells). Because 27SA 3 is the direct ...
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.
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...
2013
Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 30 end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 50 end maturation. Here, we identify Rrp17p as a previously unidentified 50 –30 exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 30 end formation. Rrp17p is required for efficient exonuclease digestion of the mature 50 ends of 5.8SS and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.
Nop5p Is a Small Nucleolar Ribonucleoprotein Component Required for Pre18 S rRNA Processing in Yeast
Journal of Biological Chemistry, 1998
We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37 °C. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A 0 and A 2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 co-immunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.
Eukaryotic Cell, 2004
Yeast Rrp5p, one of the fewtrans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A0to A2and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A3and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutantrrp5Δ6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in therrp5Δ6mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A2. Instead, the 32S precursor is processed directly at site A3, producing exclusively 2...