ABSTRACT
The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. In the yeast Saccharomyces cerevisiae, four common proteins are associated with H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p. In vitro reconstitution studies showed that four proteins also specifically interact with H/ACA snoRNAs in mammalian cell extracts. Two mammalian proteins, NAP57/dyskerin (the ortholog of Cbf5p) and hGAR1, have been characterized. In this work we describe properties of hNOP10 and hNHP2, human orthologs of yeast Nop10p and Nhp2p, respectively, and further characterize hGAR1. hNOP10 and hNHP2 complement yeast cells depleted of Nhp2p and Nop10p, respectively. Immunoprecipitation experiments with extracts from transfected HeLa cells indicated that epitope-tagged hNOP10 and hNHP2 specifically associate with hGAR1 and H/ACA RNAs; they also interact with the RNA subunit of telomerase, which contains an H/ACA-like domain in its 3' moiety. Immunofluorescence microscopy experiments showed that hGAR1, hNOP10, and hNHP2 are localized in the dense fibrillar component of the nucleolus and in Cajal (coiled) bodies. Deletion analysis of hGAR1 indicated that its evolutionarily conserved core domain contains all the signals required for localization, but progressive deletions from either the N or the C terminus of the core domain abolish localization in the nucleolus and/or the Cajal bodies.
Subject(s)
Nuclear Proteins/isolation & purification , RNA-Binding Proteins/isolation & purification , Ribonucleoproteins, Small Nucleolar/chemistry , Telomerase/chemistry , Amino Acid Sequence , Cell Cycle Proteins/metabolism , Cell Nucleolus/chemistry , Gene Deletion , Genetic Complementation Test , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Transport , RNA, Small Nucleolar/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins, Small Nuclear , Ribonucleoproteins, Small Nucleolar/genetics , Sequence Homology, Amino AcidABSTRACT
The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3'-terminal stem-loop followed by box ACA (U17/3'st) was sufficient to form an RNP, and U17/3'st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3' moiety of human telomerase RNA (hTR), and its 3'-terminal stem-loop (hTR/3'st), also could form an RNP by binding H/ACA proteins. Hence, the 3'-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins.
Subject(s)
RNA, Small Nucleolar/chemistry , RNA, Small Nucleolar/genetics , Ribonucleoproteins, Small Nuclear/genetics , Ribonucleoproteins, Small Nucleolar/metabolism , Telomerase/genetics , Amino Acid Sequence , Arginine/chemistry , Base Sequence , Binding, Competitive , Cloning, Molecular , Glycine/chemistry , HeLa Cells , Humans , Models, Genetic , Molecular Sequence Data , Plasmids , Precipitin Tests , Protein Structure, Tertiary , Transcription, GeneticABSTRACT
Small nucleolar ribonucleoprotein particles (snoRNPs) mainly catalyze the modification of rRNA. The two major classes of snoRNPs, box H/ACA and box C/D, function in the pseudouridylation and 2'-O-methylation, respectively, of specific nucleotides. The emerging view based on studies in yeast is that each class of snoRNPs is composed of a unique set of proteins. Here we present a characterization of mammalian snoRNPs. We show that the previously characterized NAP57 is specific for box H/ACA snoRNPs, whereas the newly identified NAP65, the rat homologue of yeast Nop5/58p, is a component of the box C/D class. Using coimmunoprecipitation experiments, we show that the nucleolar and coiled-body protein Nopp140 interacts with both classes of snoRNPs. This interaction is corroborated in vivo by the exclusive depletion of snoRNP proteins from nucleoli in cells transfected with a dominant negative Nopp140 construct. Interestingly, RNA polymerase I transcription is arrested in nucleoli depleted of snoRNPs, raising the possibility of a feedback mechanism between rRNA modification and transcription. Moreover, the Nopp140-snoRNP interaction appears to be conserved in yeast, because depletion of Srp40p, the yeast Nopp140 homologue, in a conditional lethal strain induces the loss of box H/ACA small nucleolar RNAs. We propose that Nopp140 functions as a chaperone of snoRNPs in yeast and vertebrate cells.
Subject(s)
Conserved Sequence , Hydro-Lyases , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Ribonucleoproteins, Small Nuclear , Ribonucleoproteins, Small Nucleolar/chemistry , Ribonucleoproteins, Small Nucleolar/metabolism , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , COS Cells , Cell Nucleolus/chemistry , Cell Nucleolus/enzymology , Cell Nucleolus/metabolism , Conserved Sequence/genetics , Epistasis, Genetic , Genetic Complementation Test , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Molecular Weight , Mutation/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Phosphoproteins/deficiency , Phosphoproteins/genetics , Protein Binding , RNA Polymerase I/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Rats , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Ribonucleoproteins, Small Nucleolar/deficiency , Ribonucleoproteins, Small Nucleolar/genetics , Saccharomyces cerevisiae/genetics , Serine-Arginine Splicing Factors , Transcription, Genetic/genetics , Transcription, Genetic/physiologyABSTRACT
Maturation of pre-ribosomal RNA (pre-rRNA) in eukaryotic cells takes place in the nucleolus and involves a large number of cleavage events, which frequently follow alternative pathways. In addition, rRNAs are extensively modified, with the methylation of the 2'-hydroxyl group of sugar residues and conversion of uridines to pseudouridines being the most frequent modifications. Both cleavage and modification reactions of pre-rRNAs are assisted by a variety of small nucleolar RNAs (snoRNAs), which function in the form of ribonucleoprotein particles (snoRNPs). The majority of snoRNAs acts as guides directing site-specific 2'-O-ribose methylation or pseudouridine formation. Over one hundred RNAs of this type have been identified to date in vertebrates and the yeast Saccharomyces cerevisiae. This number is readily explained by the findings that one snoRNA acts as a guide usually for one or at most two modifications, and human rRNAs contain 91 pseudouridines and 106 2'-O-methyl residues. In this article we review information about the biogenesis, structure and function of guide snoRNAs.
