RESUMEN

Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2'-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5' exon but also onto position 39 (universal tRNA numbering) in the 3' exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon-intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs.

Asunto(s)

ARN de Archaea/metabolismo , ARN Nucleolar Pequeño/metabolismo , ARN de Transferencia/metabolismo , Ribosa/metabolismo , Secuencia de Bases , ADN de Archaea/química , ADN de Archaea/genética , Genoma Arqueal , Intrones/genética , Metilación , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Nucleósidos/genética , Nucleósidos/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Filogenia , Plásmidos/genética , Pyrococcus/genética , Pyrococcus/metabolismo , ARN de Archaea/química , ARN de Archaea/genética , ARN Nucleolar Pequeño/genética , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia de Triptófano/genética , ARN de Transferencia de Triptófano/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico

RESUMEN

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:

Asunto(s)

Técnicas Genéticas , Ratones/genética , ARN/química , ARN/genética , Animales , Emparejamiento Base , Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario , Bases de Datos como Asunto , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Ratas

RESUMEN

Antisense box C/D small nucleolar RNAs (snoRNAs) guide the 2'-O-ribose methylations of eukaryotic rRNAs and small nuclear RNAs (snRNAs) through formation of a specific base pairing at each RNA methylation site. By analysis of a box C/D snoRNA cDNA library constructed from rat brain RNAs, we have identified a novel box C/D snoRNA, RBII-36, which is devoid of complementarity to rRNA or an snRNA and exhibits a brain-specific expression pattern. It is uniformly expressed in all major areas of adult rat brain (except for choroid plexus) and throughout rat brain ontogeny but exclusively detected in neurons in which it exhibits a nucleolar localization. In vertebrates, known methylation guide snoRNAs are intron-encoded and processed from transcripts of housekeeping genes. In contrast, RBII-36 snoRNA is intron-encoded in a gene preferentially expressed in the rat central nervous system and not in proliferating cells. Remarkably, this host gene, which encodes a previously reported noncoding RNA, Bsr, spans tandemly repeated 0.9-kilobase units including the snoRNA-containing intron. The novel brain-specific snoRNA appears to result not only from processing of the debranched lariat but also from endonucleolytic cleavages of unspliced Bsr RNA (i.e. an alternative splicing-independent pathway unreported so far for mammalian intronic snoRNAs). Sequences homologous to RBII-36 snoRNA were exclusively detected in the Rattus genus of rodents, suggesting a very recent origin of this brain-specific snoRNA.

Asunto(s)

ARN Nuclear Pequeño/genética , ARN no Traducido/genética , Animales , Secuencia de Bases , Encéfalo , Clonación Molecular , Intrones , Datos de Secuencia Molecular , Especificidad de Órganos , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/biosíntesis , Ratas , Secuencias Repetidas en Tándem/genética

RESUMEN

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.

Asunto(s)

Genoma Arqueal , Metilación , Pyrococcus/genética , ARN de Archaea/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/genética , Secuencia de Bases , Secuencia de Consenso/genética , Bases de Datos Factuales , Células Eucariotas/metabolismo , Genes Arqueales/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Mapeo Físico de Cromosoma , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN de Archaea/química , ARN de Archaea/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Nucleolar Pequeño/metabolismo , Ribosa/metabolismo , Homología de Secuencia de Ácido Nucleico , Programas Informáticos

RESUMEN

We have identified three C/D-box small nucleolar RNAs (snoRNAs) and one H/ACA-box snoRNA in mouse and human. In mice, all four snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively expressed in the brain, unlike all other known snoRNAs. Two of the human RNA orthologues (HBII-52 and HBI-36) share this expression pattern, and the remainder, HBII-13 and HBII-85, are prevalently expressed in that tissue. In mice and humans, the brain-specific H/ACA box snoRNA (MBI-36 and HBI-36, respectively) is intron-encoded in the brain-specific serotonin 2C receptor gene. The three human C/D box snoRNAs map to chromosome 15q11-q13, within a region implicated in the Prader-Willi syndrome (PWS), which is a neurogenetic disease resulting from a deficiency of paternal gene expression. Unlike other C/D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a tandemly repeated array of 47 or 24 units, respectively. In mouse the homologue of HBII-52 is processed from intronic portions of the tandem repeats. Interestingly, these snoRNAs were absent from the cortex of a patient with PWS and from a PWS mouse model, demonstrating their paternal imprinting status and pointing to their potential role in the etiology of PWS. Despite displaying hallmarks of the two families of ubiquitous snoRNAs that guide 2'-O-ribose methylation and pseudouridylation of rRNA, respectively, they lack any telltale rRNA complementarity. Instead, brain-specific C/D box snoRNA HBII-52 has an 18-nt phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA.

Asunto(s)

Encéfalo/metabolismo , Impresión Genómica , ARN Nucleolar Pequeño , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Ratas , Secuencias Repetidas en Tándem , Distribución Tisular

RESUMEN

Mechanisms of ITS2 excision from pre-rRNA remain largely elusive. In mammals, at least two endonucleolytic cleavages are involved, which result in the transient accumulation of precursors to 5.8S rRNA termed 8S and 12S RNAs. We have sequenced ITS2 in four new species of the Mus genus and investigated its secondary structure using thermodynamic prediction and comparative approach. Phylogenetic evidence supports an ITS2 folding organized in four domains of secondary structure extending from a preserved structural core. This folding is also largely conserved for the previously available mammalian ITS2 sequences, rat and human, despite their extensive sequence divergence relative to the Mus species. Conserved structural features include the structural core, containing the 3' end of 8S pre-rRNA within a single-stranded sequence, and a stem containing the 3' end of the 12S pre-rRNA species. A putative, phylogenetically preserved pseudoknot has been detected 1 nt downstream from the 12S 3' end. Two long complementarities have also been identified, in sequences conserved among vertebrates, between the pre-rRNA 32S and the snoRNA (small nucleolar RNA) U8 which is required for the excision of Xenopus ITS2. The first complementarity involves the 5.8S-ITS2 junction and 13 nt at the 5' end of U8, whereas the other one occurs between a mature 28S rRNA segment known to be required for ITS2 excision and positions 15-25 of snoRNA U8. These two potential interactions, in combination with ITS2 folding, could organize a functional pocket containing three cleavage sites and key elements for pre-rRNA processing, suggesting a chaperone role for the snoRNA U8.

Asunto(s)

ADN Ribosómico/química , Evolución Molecular , Precursores del ARN/química , ARN Nuclear Pequeño , Animales , Secuencia de Bases , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Muridae , Conformación de Ácido Nucleico , Ratas , Homología de Secuencia de Ácido Nucleico

RESUMEN

Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.

Asunto(s)

Endorribonucleasas/metabolismo , Exorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , ADN de Hongos/genética , Expresión Génica , Genes Fúngicos , Metilación , Datos de Secuencia Molecular , Familia de Multigenes , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Procesamiento Postranscripcional del ARN , ARN sin Sentido/química , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN de Hongos/química , ARN Ribosómico/biosíntesis , ARN Nuclear Pequeño/química , Ribonucleasa III , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Pequeño no Traducido

RESUMEN

The protein sequences of three known RNA 2'-O-ribose methylases were used as probes for detecting putative homologs through iterative searches of genomic databases. We have identified 45 new positive Open Reading Frames (ORFs), mostly in prokaryotic genomes. Five complete eukaryotic ORFs were also detected, among which was a single ORF (YDL112w) in the yeast Saccharomyces cerevisiae genome. After genetic depletion of YDL112w, we observed a specific defect in tRNA ribose methylation, with the complete disappearance of Gm18 in all tRNAs that naturally contain this modification, whereas other tRNA ribose methylations and the complex pattern of rRNA ribose methylations were not affected. The tRNA G18 methylation defect was suppressed by transformation of the disrupted strain with a plasmid allowing expression of YDL112wp. The formation of Gm18 on an in vitro transcript of a yeast tRNASer naturally containing this methylation, which was efficiently catalyzed by cell-free extracts from the wild-type yeast strain, did not occur with extracts from the disrupted strain. The protein encoded by the YDL112w ORF, termed Trm3 (tRNA methylation), is therefore likely to be the tRNA (Gm18) ribose methylase. In in vitro assays, its activity is strongly dependent on tRNA architecture. Trm3p, the first putative tRNA ribose methylase identified in an eukaryotic organism, is considerably larger than its Escherichia coli functional homolog spoU (1,436 amino acids vs. 229 amino acids), or any known or putative prokaryotic RNA ribose methyltransferase. Homologs found in human (TRP-185 protein), Caenorhabditis elegans and Arabidopsis thaliana also exhibit a very long N-terminal extension not related to any protein sequence in databases.

Asunto(s)

Metiltransferasas/genética , Sistemas de Lectura Abierta/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Guanosina/análogos & derivados , Guanosina/genética , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , ARN de Hongos/genética , ARN de Transferencia de Serina/genética , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Especificidad por Sustrato/genética

RESUMEN

Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs. Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells. We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide. However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex. Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.

Asunto(s)

Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/metabolismo , Animales , Composición de Base , Secuencia de Bases , Células L , Metilación , Ratones , Mutagénesis Sitio-Dirigida , Ácidos Nucleicos Heterodúplex/química , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Ribosómico/química , ARN Nuclear Pequeño/química , ARN Pequeño no Traducido

RESUMEN

Eukaryotic rRNAs contain a complex set of ribose-methylated nucleotides. Why are these nucleotides modified and how are they selected? A large family of small nucleolar RNAs (snoRNAs) with long complementarities to sites of rRNA methylation has been recently found to guide such modifications, opening up a direct approach to the study of their elusive function. Ribose methylation can also be targeted to non-rRNA sequences by tailored snoRNA guides, possibly providing a highly selective tool for altering gene expression at the post-transcriptional level.

Asunto(s)

ARN Ribosómico/metabolismo , Ribosa/metabolismo , Secuencia de Bases , Sitios de Unión , Metilación , Modelos Moleculares , Conformación de Ácido Nucleico , ARN sin Sentido , ARN Ribosómico/química

Asunto(s)

Bulgaria , Ciclo Celular/fisiología , Historia del Siglo XX , Biología Molecular/educación , Biología Molecular/historia , ARN Ribosómico/historia

RESUMEN

The first cleavage in the processing of the rRNA primary transcript in mammals occurs within the 5'-terminal region of the 5' external transcribed spacer (5'ETS), which makes the upstream portion of this spacer a selective marker of nascent transcripts. Moreover, short treatments with low doses of actinomycin D (AMD), which selectively suppress pre-rRNA synthesis and allow processing of preformed pre-rRNAs, result in the production of prematurely terminated transcripts essentially spanning the 5'ETS leader region. To gain further insight into the intranucleolar localization of early stages of preribosome formation we analyzed the distribution of this specific pre-rRNA segment by in situ hybridization at the ultrastructural level in AMD-treated or in control 3T3 mouse cells. In control cells, 5'ETS leader rRNA was detected at the border of the fibrillar centers and over the dense fibrillar component, in agreement with previous data suggesting that rRNA gene transcription takes place at the border of the fibrillar centers before a rapid transfer of the nascent trancript to the dense fibrillar component. Observation of cells subjected to a short treatment with low doses of AMD fully supports this conclusion, with the prematurely terminated 5'ETS leader-containing transcripts detected at the border of enlarged fibrillar centers. With prolonged periods of AMD treatment even the partial transcription of rRNA genes is blocked and fibrillar centers of typically segregated nucleoli show no positive signals with the 5'ETS leader probe. We also analyzed in parallel the intranucleolar distribution of U3 small nucleolar RNA, which is involved in 5'ETS processing, by hybridization with biotinylated antisense oligonucleotides. Distribution of U3 roughly paralleled that of 5'ETS leader rRNA in untreated cells. However, U3 RNA persisted in the dense fibrillar component of segregated nucleoli whatever the conditions of drug treatment, i.e., even after a thorough chase of the rRNA precursors from this nucleolar compartment.

Asunto(s)

Nucléolo Celular/ultraestructura , Hibridación in Situ/métodos , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Ribosómico/genética , Células 3T3/efectos de los fármacos , Animales , Biotina/química , Nucléolo Celular/genética , Sondas de ADN/química , Sondas de ADN/genética , Dactinomicina/farmacología , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Precursores del ARN/genética , ARN Mensajero/química , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo

RESUMEN

Molecular mechanisms involved in the nucleolytic cleavage at the 18S rRNA/internal transcribed spacer 1 (ITS 1) junction, a late step of small-subunit pre-rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful in vitro assays. To identify the minimal cis-acting signals required for this reaction, we studied the processing of truncated human rRNA gene transcripts transiently expressed upon transfection of rRNA minigenes into cultured mouse cells. We observed that processing at this site was faithfully reproduced with transcripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by further shortening of either sequence. Remarkably, this minimal transcript contains, within its 18S rRNA part, long sequences complementary to both U20 and U13 small nucleolar RNAs (snoRNAs). The cis-acting elements essential for the reaction were studied further by site-directed mutagenesis. The U20 snoRNA complementary region in 18S rRNA was not required for faithful processing at the 18S rRNA/ITS 1 junction. Also, processing at this site was not appreciably altered by random substitution of proximal ITS 1 sequences (including the 5' terminal nucleotide) or of the terminal nucleotide of mature 18S rRNA. Substitutions in the four-nucleotide loop of the 18S rRNA 3'-terminal stem-loop, including the two adenosine residues substrates of dimethylation, did not alter appreciably the formation of the 18S rRNA 3' end, showing that the (methyl)2A1850.(methyl)2A1851 doublet was not required for processing at this site. Two highly conserved 18S rRNA elements acted as major cis-acting signals for processing at the 3' end, the CAUU sequence immediately preceding the 3'-terminal nucleotide and the 3' strand of the 3'-terminal 18S rRNA helix, complementary to U13 snoRNA. Compensatory mutations, restoring the potential for helix formation, but not U13 snoRNA complementarity, did not restitute the cleavage at the 3' end of 18S rRNA. This suggests that U13 snoRNA may be a trans-acting factor in the nucleolytic cleavage at the 3' end of 18S rRNA.

Asunto(s)

Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico 18S/química , ARN Ribosómico 18S/metabolismo , ARN Nuclear Pequeño/metabolismo , Animales , Secuencia de Bases , Células L , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , ARN Ribosómico 18S/biosíntesis , Transcripción Genética , Transfección

RESUMEN

Eukaryotic ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides. This takes place in the nucleolus and involves a family of small nucleolar RNAs (snoRNAs) with long regions (10-21 nucleotides) complementary to rRNA sequences spanning the methylation site--a complementary snoRNA is required for methylation at a specific site. Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identified structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when coexpressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool for the alteration of gene expression at the post-transcriptional level.

Asunto(s)

ARN sin Sentido/metabolismo , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribosa/metabolismo , Animales , Composición de Base , Línea Celular , Células HeLa , Humanos , Metilación , Ratones , Mutación , Conformación de Ácido Nucleico , ARN Ribosómico/genética

RESUMEN

A growing number of small nucleolar RNAs (snoRNAs) are intron-encoded, contain the characteristic box C (UGAUGA) and box D (CUGA) motifs and exhibit long complementarities to conserved sequences in mature rRNAs. We have identified nine additional members of this family, U32 to U40. All but one are encoded in introns of ribosomal protein genes in vertebrates: U32 to U35 in rpL13a, U36 in rpL7a and U38 to U40 in rpS8. By contrast, U37 is encoded in elongation factor 2 gene. Interestingly, U32 and U36 each contain two complementarities (one to 18 S and the other to 28 S rRNA). U32 to U40 are fibrillarin-associated, devoid of a 5'-trimethyl-cap and display an exclusively nucleolar localization. They are all metabolically stable and roughly as abundant as previously reported members of this family. Characterization of their homologs in distant species shows that their 10 to 14 nt long rRNA complementarities are conserved. A clue on the function of this snoRNA family is provided by the comparative analysis of the largely expanded collection of their conserved duplexes with rRNA. Not only does each duplex span at least one site of 2'-O-ribose methylation in the rRNA but the modification site is always at the same position in the duplex, paired to the fifth nucleotide upstream from a box D motif in the snoRNA. Consistent with the notion that each snoRNA of this family guides one particular methylation along the rRNA sequence, we have detected several pairs of snoRNAs with overlapping complementarities to rRNA tracts with vicinal sites of ribose methylations. In each case, the two overlapping complementarities are shifted from each other by a distance equal to the spacing between the methylated sites which are thus found at the same position within each of the mutually exclusive duplexes. Finally, we have also identified, within three previously known snoRNAs, novel antisense elements able to form a canonical duplex around ribose-methylated sites in rRNA, which further supports the conclusion that the duplex structure provides the 2'-O-methyltransferase with the appropriate site-specificity on the substrate.

Asunto(s)

Nucléolo Celular/química , Intrones/genética , Procesamiento Postranscripcional del ARN , ARN sin Sentido/metabolismo , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Proteínas Cromosómicas no Histona/metabolismo , Secuencia Conservada , Bases de Datos Factuales , Evolución Molecular , Humanos , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Precursores del ARN/metabolismo , ARN sin Sentido/química , ARN sin Sentido/genética , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Ribosa/metabolismo

RESUMEN

Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs. Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2'-O-methylation of preribosomal RNA (pre-rRNA). Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA. The antisense element, together with the D or D' box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction. The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated.

Asunto(s)

Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/fisiología , Ribosa/genética , Secuencia de Bases , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Secuencia de Consenso , Células HeLa/fisiología , Humanos , Metilación , Datos de Secuencia Molecular , Nucleótidos/metabolismo , ARN sin Sentido/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribosa/metabolismo , Levaduras/genética , ARNt Metiltransferasas/metabolismo

RESUMEN

U21 is an intron-encoded snoRNA in vertebrates which contains a 13-nt tract of complementarity to an invariant sequence in eukaryotic 28S rRNA. Here, we report the characterization of its Drosophila melanogaster homolog which is the first case of an intron-encoded snoRNA in an invertebrate metazoan. In D. melanogaster, U21 is encoded within the ARF-1 (ADP ribosylation factor 1) gene, whereas in chicken and mammals it is found in the ribosomal protein L5 gene. In D. melanogaster, like in vertebrates, U21 is devoid of a 5' trimethylguanosine cap, thus, likely resulting from processing of intronic RNA. The only portion of U21 sequence preserved between D. melanogaster and vertebrates, in addition to the hallmark box C and box D motifs, corresponds precisely to the 13-nt complementary to rRNA, pointing to an important role of the pairing of U21 to pre-rRNA.

Asunto(s)

ARN Ribosómico 28S/genética , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Pollos/genética , ADN Complementario , Drosophila melanogaster/genética , Humanos , Intrones , Datos de Secuencia Molecular , Procesamiento Postranscripcional del ARN , ARN Mensajero , ARN Ribosómico 28S/química , ARN Nuclear Pequeño/química , Homología de Secuencia de Ácido Nucleico

RESUMEN

Nucleoli contain complex populations of small nucleolar RNAs (snoRNAs) likely to be involved in pre-rRNA processing and ribosome biogenesis. A growing family of snoRNAs which interacts with nucleolar protein fibrillarin is structurally related by the presence of long complementarities to rRNA (12 to 21 nucleotides) and of a pair of common sequence motifs, termed boxes C and D. All are encoded in introns and produced by processing of intronic RNA. We have analysed the mechanism of processing of one of these snoRNAs, U20, by transfection in mouse cells. We show here that the cis-acting signals for its processing are restricted to a minor portion of the mature snoRNA sequence. A terminal structure in which the two box motifs are brought in close vicinity by the pairing of the 5' and 3' terminal nucleotides is sufficient to direct faithful processing. Particularly, the key role of the terminal stem shared by most snoRNAs of this family is demonstrated by the effect of compensatory mutations. Our results also indicate that faithful processing at both ends of the snoRNA can be uncoupled and that it is not strictly dependent on pre-mRNA splicing. Finally, our data point to the exclusive involvement of 5'-->3' and 3'-->5' exonucleolytic activities in the processing of intronic snoRNAs of this family.

Asunto(s)

Proteínas Cromosómicas no Histona/metabolismo , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Clonación Molecular , Secuencia Conservada/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Fosfoproteínas/genética , Plásmidos/genética , Caperuzas de ARN/genética , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , Transcripción Genética/genética , Transfección/genética , Nucleolina

RESUMEN

Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.

Asunto(s)

Nucléolo Celular/genética , Intrones , ARN Ribosómico/genética , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Código Genético , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico

RESUMEN

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Asunto(s)

ARN Ribosómico 28S/genética , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Nucléolo Celular/metabolismo , Pollos , Secuencia Conservada , ADN Complementario/genética , Genes Fúngicos , Células HeLa , Humanos , Intrones , Datos de Secuencia Molecular , Filogenia , ARN de Hongos/genética , Saccharomyces cerevisiae/genética