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1.
Nucleic Acids Res ; 48(21): 12310-12325, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33166396

RESUMO

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.


Assuntos
Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Biogênese de Organelas , Fatores de Terminação de Peptídeos/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , tRNA Metiltransferases/genética , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , RNA Ribossômico 5,8S/biossíntese , RNA Ribossômico 5,8S/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
2.
RNA ; 21(7): 1240-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25995445

RESUMO

Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.


Assuntos
RNA Ribossômico 5,8S/biossíntese , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Animais , Exorribonucleases/genética , Técnicas de Silenciamento de Genes , Camundongos , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA
3.
FEBS J ; 275(16): 4164-78, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18631361

RESUMO

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Deltanop53/GAL::NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 5,8S/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Nucléolo Celular/química , DNA Polimerase Dirigida por DNA/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Teste de Complementação Genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poliadenilação , Precursores de RNA/biossíntese , RNA Ribossômico 5,8S/biossíntese , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Transcrição Gênica
4.
J Cell Physiol ; 214(2): 381-8, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17654514

RESUMO

Ribosomal RNA (rRNA) in vertebrates is initially transcribed as a single 47S precursor which is modified by the addition of 2'-O-methyl ribose moieties, pseudouridines, and methyl groups, followed by cleavage at several sites to produce the mature 28S, 18S, and 5.8S rRNAs. Cleavage of the rRNA precursor to generate the 18S rRNA is mediated by a ribonucleoprotein (RNP) complex termed the processome containing U3, a box C/D small nucleolar RNA (snoRNA), and at least 28 cellular proteins. We previously identified a novel human RNA binding protein, NF-kappaB binding protein (NFBP), which is the human homolog of Rrp5p, a protein component of the yeast U3 processome. Here, we show that NFBP colocalizes with and coprecipitates U3 in the nucleolus. We also demonstrate that NFBP is essential for the generation of 18S rRNA as maturation of the 18S rRNA is repressed in the absence of NFBP. Using Northern blot analyses, we further show that NFBP is specifically necessary for cleavages at sites A0, 1, and 2, as unprocessed intermediate forms of rRNA accumulated in the absence of NFBP.


Assuntos
Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Hibridização In Situ , Antígenos de Histocompatibilidade Menor , Testes de Precipitina , RNA Ribossômico 18S/biossíntese , RNA Ribossômico 28S/biossíntese , RNA Ribossômico 5,8S/biossíntese , RNA Interferente Pequeno/metabolismo , Transfecção
5.
Mol Cell ; 9(2): 341-51, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11864607

RESUMO

Ssf1p and Ssf2p are two nearly identical and functionally redundant nucleolar proteins. In the absence of Ssf1p and Ssf2p, the 27SA(2) pre-rRNA was prematurely cleaved, inhibiting synthesis of the 27SB and 7S pre-rRNAs and the 5.8S and 25S rRNA components of the large ribosomal subunit. On sucrose gradients, Ssf1p sedimented with pre-60S ribosomal particles. The 27SA(2), 27SA(3), and 27SB pre-rRNAs were copurified with tagged Ssf1p, as were 23 large subunit ribosomal proteins and 21 other proteins implicated in ribosome biogenesis. These included four Brix family proteins, Ssf1p, Rpf1p, Rpf2p, and Brx1p, indicating that the entire family functions in ribosome synthesis. This complex is distinct from recently reported pre-60S complexes in RNA and protein composition. We describe a multistep pathway of 60S preribosome maturation.


Assuntos
Proteínas Nucleares/fisiologia , Precursores de RNA/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Centrifugação com Gradiente de Concentração , Sequência Consenso , Deleção de Genes , Substâncias Macromoleculares , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/biossíntese , RNA Ribossômico 5,8S/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
6.
RNA ; 7(9): 1317-34, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11565753

RESUMO

Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas , RNA Helicases/fisiologia , RNA Nucleotidiltransferases , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Nucléolo Celular/metabolismo , RNA Helicases DEAD-box , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polirribossomos , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico 5,8S/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Técnicas do Sistema de Duplo-Híbrido
7.
RNA ; 5(6): 779-93, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10376877

RESUMO

The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.


Assuntos
Proteínas Nucleares/fisiologia , RNA Fúngico/metabolismo , RNA Ribossômico 18S/biossíntese , RNA Ribossômico 5,8S/biossíntese , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Sobrevivência Celular/genética , Deleção de Genes , Genes/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação Proteica , Precursores de RNA/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 5,8S/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequências de Repetição em Tandem , Temperatura , Transativadores/metabolismo
8.
RNA ; 4(10): 1268-81, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9769101

RESUMO

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.


Assuntos
Polirribossomos/metabolismo , RNA Nucleotidiltransferases/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Nucléolo Celular/química , RNA Helicases DEAD-box , RNA Nucleotidiltransferases/análise , RNA Nucleotidiltransferases/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/biossíntese , RNA Ribossômico 5,8S/biossíntese , Proteínas Recombinantes de Fusão
9.
RNA ; 4(5): 566-81, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9582098

RESUMO

Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit.


Assuntos
RNA Helicases , RNA Nucleotidiltransferases/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Nucléolo Celular/química , Clonagem Molecular , RNA Helicases DEAD-box , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta/genética , RNA Nucleotidiltransferases/análise , RNA Nucleotidiltransferases/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/biossíntese , RNA Ribossômico 5,8S/biossíntese , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento
10.
RNA ; 3(5): 476-88, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9149229

RESUMO

The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation.


Assuntos
Variação Genética , RNA Ribossômico 5,8S/biossíntese , RNA Ribossômico/biossíntese , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Escherichia coli , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/química , RNA Ribossômico 5,8S/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
11.
RNA ; 3(4): 382-91, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9085845

RESUMO

We have isolated suppressors of the temperature-sensitive rRNA processing mutation rrp2-2 in Saccharomyces cerevisiae. A class of extragenic suppressors was mapped to the YBR257w reading frame in the right arm of Chromosome II. Characterization of this gene, renamed POP4, shows that the gene product is necessary both for normal 5.8S rRNA processing and for processing of tRNA. Immunoprecipitation studies indicate that Pop4p is associated with both RNase MRP and RNase P. The protein is also required for accumulation of RNA from each of the two ribonucleoprotein particles.


Assuntos
Endorribonucleases/genética , Processamento Pós-Transcricional do RNA , RNA Catalítico/genética , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , RNA Ribossômico 5,8S/biossíntese , RNA de Transferência/biossíntese , Ribonuclease P , Saccharomyces cerevisiae/enzimologia
12.
EMBO J ; 15(20): 5701-14, 1996 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8896463

RESUMO

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S) are synthesized as a single precursor which is subsequently processed into the mature rRNAs by a complex series of cleavage and modification reactions. In the yeast Saccharomyces cerevisiae, the early pre-rRNA cleavages at sites A0, A1 and A2, required for the synthesis of 18S rRNA, are inhibited in strains lacking RNA or protein components of the U3, U14, snR10 and snR30 small nucleolar ribonucleoproteins (snoRNPs). The subsequent cleavage at site A3, required for formation of the major, short form of 5.8S rRNA, is carried out by another ribonucleoprotein, RNase MRP. A screen for mutations showing synthetic lethality with deletion of the non-essential snoRNA, snR10, identified a novel gene, RRP5, which is essential for viability and encodes a 193 kDa nucleolar protein. Genetic depletion of Rrp5p inhibits the synthesis of 18S rRNA and, unexpectedly, also of the major short form of 5.8S rRNA. Pre-rRNA processing is concomitantly impaired at sites A0, A1, A2 and A3. This distinctive phenotype makes Rrp5p the first cellular component simultaneously required for the snoRNP-dependent cleavage at sites A0, A1 and A2 and the RNase MRP-dependent cleavage at A3 and provides evidence for a close interconnection between these processing events. Putative RRP5 homologues from Caenorhabditis elegans and humans were also identified, suggesting that the critical function of Rrp5p is evolutionarily conserved.


Assuntos
Proteínas Nucleares/química , RNA Fúngico/biossíntese , RNA Ribossômico 18S/biossíntese , RNA Ribossômico 5,8S/biossíntese , Proteínas de Saccharomyces cerevisiae , Alelos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Nucléolo Celular/metabolismo , Sobrevivência Celular , Humanos , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae , Alinhamento de Sequência , Proteína Estafilocócica A/metabolismo
13.
Mol Biochem Parasitol ; 72(1-2): 227-37, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8538692

RESUMO

Here we present the sequence of the large subunit (LSU) rRNA expressed in blood-stage forms (and therefore A-type) of the malaria parasite, Plasmodium falciparum, from two different isolates. We determined the genomic sequence of a rRNA unit of the CAMP parasite strain from within the internal transcribed spacer 1 (ITS1) through the 5.8S rRNA gene, the ITS2 and the entire large subunit rRNA gene. We have also determined the corresponding sequence of the gene of the FVO strain by sequencing cDNA clones derived from blood-stage asexual parasites. Differences between the two were due to scattered point mutations in expansion segments of the mature rRNA regions. In addition to the point mutations, the rRNA genes from the two strains could be distinguished by the presence of a more complex polymorphism near the 3' end of the molecule. The most complex polymorphic form was localized on a single chromosome and found in only a subset of geographically distinct isolates. The sequences of the 5.8S rRNA unit and the LSU rRNA unit reported here can be logically assembled into a complete secondary structure which conforms to the standard structure conserved in all eukaryotic ribosomes. The construction of a model of secondary structure for the LSU rRNA has allowed the identification of phylogenetically conserved sequences involved in drug interaction with the ribosome, as well as those sequences involved in tertiary interactions within the rRNA itself.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários , Conformação de Ácido Nucleico , Plasmodium falciparum/metabolismo , RNA de Protozoário/química , RNA Ribossômico 5,8S/química , RNA Ribossômico/química , Animais , Sequência de Bases , DNA de Protozoário/genética , DNA Ribossômico/genética , Resistência Microbiana a Medicamentos , Dados de Sequência Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , RNA de Protozoário/biossíntese , RNA de Protozoário/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , RNA Ribossômico 5,8S/biossíntese , RNA Ribossômico 5,8S/genética , Ribossomos/efeitos dos fármacos
14.
Biochem Biophys Res Commun ; 202(1): 225-33, 1994 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-7518675

RESUMO

The RNA component of the mitochondrial RNA processing (MRP) enzyme is related to both replication of mitochondrial DNA and processing of 5.8S rRNA, which are accelerated in the frog earliest stage (stage I) of frog oocytes. Microinjection of the deleted genes into the stage I oocytes showed positive cis-elements in the upstream region of the gene. The specific binding of protein(s) to this region was detected in cell extracts from stage I oocytes and liver but not in extracts of stage II-IV oocytes and the concentration of this protein was 40 times higher in the extract of stage I oocytes than that in liver.


Assuntos
Endorribonucleases/biossíntese , Regulação Enzimológica da Expressão Gênica , Oócitos/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Cromatografia de Afinidade , DNA/química , DNA/metabolismo , Primers do DNA , Replicação do DNA , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/genética , Feminino , Deleção de Genes , Fígado/enzimologia , Dados de Sequência Molecular , Oócitos/enzimologia , Reação em Cadeia da Polimerase/métodos , RNA/biossíntese , RNA Mitocondrial , RNA Ribossômico 5,8S/biossíntese , Ranidae , Mapeamento por Restrição , Sefarose/análogos & derivados , Xenopus laevis
15.
EMBO J ; 13(10): 2452-63, 1994 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-7515008

RESUMO

We have developed techniques for the detailed analysis of cis-acting sequences in the pre-rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5' end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5' to the end of 5.8S(S), and acts as an entry site for 5'-->3' exonuclease digestion which generates the 5' end of 5.8S(S). This pathway is inhibited in strains mutant for XRN1p and RAT1p. Both of these proteins have been reported to have 5'-->3' exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RAT1p and XRN1p in trans, and is kinetically faster than 5.8S(S) synthesis.


Assuntos
Exonucleases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 5,8S/biossíntese , Saccharomyces cerevisiae/genética , Sequência de Bases , Análise Mutacional de DNA , DNA Ribossômico/genética , Variação Genética , Dados de Sequência Molecular , RNA/biossíntese , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Ribossômico 5,8S/genética , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência
16.
Nucleic Acids Res ; 18(14): 4179-84, 1990 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-2198535

RESUMO

In the yeast Saccharomyces cerevisiae, each of the tandemly repeated ribosomal RNA genes carries a 5S gene within the 'non-transcribed' spacer region. These 5S RNA genes lie between the rDNA enhancer and the promoter of rRNA transcription. Since there is roughly equimolar synthesis of 5S RNA and the 35S rRNA precursor transcript we asked whether the enhancer plays a role in regulating the transcription of 5S RNA. A marked 5S gene was inserted into plasmids designed to test rDNA enhancer function. The enhancer failed to stimulate 5S RNA synthesis even though it stimulated transcription of a distal rRNA test gene greater than 10-fold. This failure is consistent with a model of enhancer function that proposes specific interactions between the enhancer and the 35S rRNA promoter via a looping out of the intervening 5S RNA gene.


Assuntos
DNA Ribossômico/genética , Elementos Facilitadores Genéticos , Regulação Fúngica da Expressão Gênica , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Fúngico/genética , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Plasmídeos , RNA Fúngico/genética , RNA Ribossômico 5,8S/biossíntese , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5S/biossíntese , Transcrição Gênica
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