Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10.

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.

A nucleolar protein related to ribosomal protein L7 is required for an early step in large ribosomal subunit biogenesis.

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 precursor to 27SB(S), we conclude that Rlp7p is specifically required for the 5' to 3' exonucleolytic trimming of the 27SA(3) into the 27SB(S) precursor. As it is essential for processing in both the major and minor pathways, we propose that Rlp7p may act as a specificity factor that binds precursor rRNAs and tethers the enzymes that carry out the early 5' to 3' exonucleolytic reactions that generate the mature rRNAs. Rlp7p may also be required for the endonucleolytic cleavage in internal transcribed spacer 2 that separates the 5.8S rRNA from the 25S rRNA.

In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs.

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.

Conserved composition of mammalian box H/ACA and box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140.

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.

Structure and biogenesis of small nucleolar RNAs acting as guides for ribosomal RNA modification.

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.

Nucleolar localization signals of box H/ACA small nucleolar RNAs.

The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3' hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5' or 3' hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.

Mutations of non-canonical base-pairs in the 3' major domain of Escherichia coli 16 S ribosomal RNA affect the initiation and elongation of protein synthesis.

Three single-point mutations and two multibase substitutions were introduced into the lower half of the 3' major domain of Escherichia coli 16 S ribosomal RNA. The three single mutations were located in helix 29 (U1341C) or in helix 43 (U1351C or A1357C) and replaced highly conserved non-canonical base-pairs with Watson-Crick base-pairs. The two multibase substitutions were located at the base of helix 42, where they transformed an irregular portion into a Watson-Crick [correction of Waston-Crick] segment. Each of the single mutations could be expressed in vivo from the rrnB operon of a multicopy plasmid under control of constitutive promoters, and none of them affected growth-rate. However, mutation A1357C, but not U1341C or U1351C, severely retarded cell growth, when expressed together with another mutation in the upper half of the 3' major domain, C1192U. The latter mutation is located in helix 34 and abolishes the binding of spectinomycin, a protein synthesis inhibitor. The proportion of mutated ribosomes was high in polysomes, suggesting that the A1357C and C1192U double mutation did not affect the initiation but rather the elongation of protein synthesis. The effect of the double mutation reveals a functional interplay between helices 34 and 43. Furthermore, an interaction between helices 34 and 43 was also suggested by studies of protection by spectinomycin against chemical attack, that showed that the binding site of spectinomycin was restored with ribosomes bearing another double mutation, U1351C and C1192U. In contrast to the single mutations, the multiple mutations in helix 42 could not be expressed in vivo under control of the strong constitutive promoters, but could be expressed under control of the weaker, thermoinducible lambda PL promoter. They did not affect cell growth, whether expressed in the absence or the presence of the C1192U mutation. However, under conditions where protein synthesis depended exclusively on ribosomes with plasmid-encoded rRNA, cells transformed with plasmids altered in helix 42 could not grow. Analysis of the plasmid-borne 16 S rRNA distribution in bacteria transformed with these mutant plasmids showed that mutant 16 S rRNA was present in a high proportion in the free 30 S subunits but was underrepresented in 70 S ribosomes and polysomes. Extension inhibition assays (toeprinting) demonstrated that this altered distribution resulted from an impaired capacity of the mutant 30 S subunits to form translation initiation complexes.

Pleiotropic effects of mutations at positions 13 and 914 in Escherichia coli 16S ribosomal RNA.

Mutations at position 13 or 914 of Escherichia coli 16S ribosomal RNA exert pleiotropic effects on protein synthesis. They interfere with the binding of streptomycin, a translational miscoding drug, to the ribosomes. They increase translational fidelity, and this effect can be related to a perturbation of the higher order structure of the 530 stem-loop, a key region for tRNA selection. In contrast, the structure of the decoding center is not perturbed. The mutations also affect translational initiation, slowing down the formation of the 30S initiation complex. This effect can be related to a destabilization of the pseudoknot helix (17-19/916-918), at the convergence of the three major domains of 16S ribosomal RNA.

Mutational and structural analysis of the RNA binding site for Escherichia coli ribosomal protein S7.

Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3' major domain of E. coli 16 S rRNA. This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G.U pair. Two hairpins with non-canonical base-pairs, 42' and 43, protrude from loops A and B, respectively. We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding. Changing the stem of hairpin 42' into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42' do not provide recognition features for S7. However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A. Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was found to be required for proper S7 binding. Moreover, replacing the U.G pair with a C.G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U.G pair is a recognition signal for S7. S7 binding was also affected by mutations in helix 29. Insertion of one nucleotide 5' to the G or 3' to the U of the G.U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G.U pair by a G.C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C.G pair upstream from the G.U pair had no effect. Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining part of loop B.

Interaction of Escherichia coli ribosomal protein S7 with 16S rRNA.

The interaction between Escherichia coli ribosomal protein S7 and 16S rRNA was investigated using in vitro synthesized RNA transcripts. It was shown by nitrocellulose membrane filtration that RNA transcripts corresponding to the 3' major domain (nucleotides 926-1393) and to the lower half of this domain (nucleotides 926-986/1219-1393) bound S7 with the same affinity as 16S rRNA. A series of deletion mutants of the DNA coding for the lower half of the 3' major domain were constructed and the corresponding RNA fragments were assayed for their capacity to bind S7. A minimal domain of 108 nucleotides which can still efficiently bind S7 was thus obtained. In this domain, the 1304-1308/1329-1333 irregular helix and the 1351-1371 irregular hairpin were found to contain important determinants for S7 binding.