Identification of novel proteins associated with yeast snR30 small nucleolar RNA (original) (raw)
Related papers
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.
Molecular and Cellular Biology, 2008
Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.
GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast
The EMBO journal, 1992
Among the few proteins of the eukaryotic nucleolus that have been characterized, four proteins, nucleolin, fibrillarin, SSB1 and NSR1, possess a common structural motif, the GAR domain, which is rich in glycine and arginine residues. In order to examine whether the presence of this domain is characteristic of a family of nucleolar proteins, we investigated whether other yeast genes encode proteins containing GAR domains. We report here the sequence and the characterization of a new yeast gene, GAR1, which encodes a protein of 205 residues containing two GAR domains. GAR1 is a non-ribosomal protein, localized in the yeast nucleolus, which is essential for cell growth. Immunoprecipitation with anti-GAR1 antibodies shows that GAR1 is associated with a subset of snoRNAs, including snR10 and snR30. Depletion of GAR1 by expression under the control of a regulated GAL promoter, impairs processing of the 35S primary transcript of pre-rRNA and prevents synthesis of 18S rRNA. GAR1 is thus the...
Molecular and Cellular Biology, 1997
We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae. The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation. To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search. The yeast Mpp10p homolog is 30% identical to the human protein over its length. Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast. As a first step in the genetic analysis of Mpp10p function, diploid S. cerevisiae cells were transformed with a null allele. Sporulation and tetrad analysis indicate that MPP10 is an essential gene. A strain was constructed where Mpp10p is expressed from a galactose-ind...
The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae
RNA, 2005
Conversion of uridines into pseudouridines (Cs) is the most frequent base modification in ribosomal RNAs (rRNAs). In eukaryotes, the pseudouridylation sites are specified by base-pairing with specific target sequences within H/ACA small nucleolar RNAs (snoRNAs). The yeast rRNAs harbor 44 Cs, but, when this work began, 15 Cs had completely unknown guide snoRNAs. This suggested that many snoRNAs remained to be discovered. To address this problem and further complete the snoRNA assignment to C sites, we identified the complete set of RNAs associated with the H/ACA snoRNP specific proteins Gar1p and Nhp2p by coupling TAP-tag purifications with genomic DNA microarrays experiments. Surprisingly, while we identified all the previously known H/ACA snoRNAs, we selected only three new snoRNAs. This suggested that most of the missing C guides were present in previously known snoRNAs but had been overlooked. We confirmed this hypothesis by systematically investigating the role of previously known, as well as of the newly identified snoRNAs, in specifying rRNA C sites and found all but one missing guide RNAs. During the completion of this work, another study, based on bioinformatic predictions, also reported the identification of most missing guide RNAs. Altogether, all C guides are now identified and we can tell that, in budding yeast, the 44 Cs are guided by 28 snoRNAs. Finally, aside from snR30, an atypical small RNA of heterogeneous length and at least one mRNA, all Gar1p and Nhp2p associated RNAs characterized by our work turned out to be snoRNAs involved in rRNA C specification.
Nucleic Acids Research, 2012
Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co-and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.
A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis
Nature, 2002
Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago 1,2 , its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested 3,4 . Biochemical and genetic analyses suggest that U3-pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages 3 . Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1-17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5 0 ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.
Febs Journal, 2005
The factors involved in rRNA processing in eukaryotes assemble cotranscriptionally onto the nascent pre-rRNAs and include endonucleases, exonucleases, RNA helicases, GTPases, modifying enzymes and snoRNPs (small nucleolar ribonucleoproteins). The precursor of three of the four eukaryotic mature rRNAs contains the rRNA sequences flanked by two internal (ITS1 and ITS2) and two external (5¢-ETS and 3¢-ETS) spacer sequences that are removed during processing . The pre-rRNA is first assembled into a 90S particle that contains U3 snoRNP and 40S subunit-processing factors . The early pre-rRNA endonucleolytic cleavages at sites A 0 , A 1 and A 2 occur within the 90S particles . A 2 cleavage releases the first pre60S particle, which differs in composition from the known 90S particle. Pre60S particles contain 27S rRNA, ribosomal L proteins and many nonribosomal proteins [6].