RESUMO
The spliceosome catalyses the splicing of nuclear pre-mRNA (precursor mRNA) in eukaryotes. Pre-mRNA splicing is essential to remove internal non-coding regions of pre-mRNA (introns) and to join the remaining segments (exons) into mRNA before translation. The spliceosome is a complex assembly of five RNAs (U1, U2, U4, U5 and U6) and many dozens of associated proteins. Although a high-resolution structure of the spliceosome is not yet available, inroads have been made towards understanding its structure and function. There is growing evidence suggesting that U2 and U6 RNAs, of the five, may contribute to the catalysis of pre-mRNA splicing. In this review, recent progress towards understanding the structure and function of U2 and U6 RNAs is summarized.
Assuntos
Domínio Catalítico , Spliceossomos/química , Spliceossomos/metabolismo , Domínio Catalítico/efeitos dos fármacos , Humanos , Metais/química , Metais/farmacologia , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Spliceossomos/genéticaRESUMO
A eukaryotic chromosome contains many genes, each transcribed separately by RNA polymerase (pol) I, II or III. Transcription termination between genes prevents the formation of polycistronic RNAs and anti-sense RNAs, which are generally detrimental to the correct expression of genes. Terminating the transcription of protein-coding genes by pol II requires a group of proteins that also direct cleavage and polyadenylation of the messenger RNA in response to a specific sequence element, and are associated with the carboxyl-terminal domain of the largest subunit of pol II (refs 1, 2, 3, 4, 5, 6). By contrast, the cis-acting elements and trans-acting factors that direct termination of non-polyadenylated transcripts made by pol II, including small nucleolar and small nuclear RNAs, are not known. Here we show that read-through transcription from yeast small nucleolar RNA and small nuclear RNA genes into adjacent genes is prevented by a cis-acting element that is recognized, in part, by the essential RNA-binding protein Nrd1. The RNA-binding protein Nab3, the putative RNA helicase Sen1, and the intact C-terminal domain of pol II are also required for efficient response to the element. The same proteins are required for maintaining normal levels of Nrd1 mRNA, indicating that these proteins may control elongation of a subset of mRNA transcripts.
Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Poli A/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/metabolismo , Transcrição Gênica , Leveduras/genética , Leveduras/metabolismoRESUMO
The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-Delta42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Delta42 is much more sensitive to mutations in a (dT-dA)(7) tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)(7) tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.
Assuntos
Cromatina/ultraestrutura , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase III/fisiologia , RNA Nuclear Pequeno/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência Rica em At , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Genes Fúngicos , Proteínas HMGN , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , RNA Nuclear Pequeno/biossíntese , Elementos de Resposta , Saccharomyces cerevisiae/metabolismo , TATA Box , Fator de Transcrição TFIIIB , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFIII/fisiologia , Transcrição GênicaRESUMO
The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Mutação , RNA Nuclear Pequeno/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Supressão Genética , Sequência de Aminoácidos , Temperatura Baixa , Sequência Conservada , Fator de Iniciação 4E em Eucariotos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Plasmídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Helicases , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Homologia de Sequência de Aminoácidos , Temperatura , Técnicas do Sistema de Duplo-HíbridoRESUMO
Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.
Assuntos
RNA Polimerase II/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alelos , Sequência de Bases , Primers do DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Supressores , Ribonucleoproteínas Nucleares Heterogêneas , Fosforilação , Testes de Precipitina , Ligação Proteica , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteínas/genética , Temperatura , Transcrição GênicaRESUMO
Primary transcripts made by RNA polymerase II (Pol II), but not Pol I or Pol III, are modified by addition of a 7-methylguanosine (m7G) residue to the triphosphate 5' end shortly after it emerges from the polymerase. The m7G "caps" of small nuclear and small nucleolar RNAs, but not messenger RNAs, are subsequently hypermethylated to a 2,2,7-trimethylguanosine (TMG) residue. U6 RNA, the only small nuclear RNA synthesized by Pol III in most eukaryotes, does not receive a methylguanosine cap. However, human U6 RNA is O-methylated on the 5'-terminal (gamma) phosphate by an enzyme that recognizes the 5' stem-loop of U6. Here we show that variant yeast U6 RNAs truncated or substituted within the 5' stem-loop are TMG capped in vivo. Accumulation of the most efficiently TMG-capped U6 RNA variant is strongly inhibited by a conditional mutation in the largest subunit of Pol III, confirming that it is indeed synthesized by Pol III. Thus, methylguanosine capping and cap hypermethylation are not exclusive to Pol II transcripts in yeast. We propose that TMG capping of variant U6 RNAs occurs posttranscriptionally due to exposure of the 5' triphosphate by disruption of protein binding and/or gamma-methyl phosphate capping. 5' truncation and TMG capping of U6 RNA does not appear to affect its normal function in splicing, suggesting that assembly and action of the spliceosome is not very sensitive to the 5' end structure of U6 RNA.
Assuntos
Proteínas Fúngicas/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Conformação de Ácido Nucleico , Capuzes de RNA/metabolismo , RNA Polimerase III/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/química , RNA Nuclear Pequeno/química , Saccharomyces cerevisiae/genética , Animais , Sequência de Bases , Proteínas Fúngicas/genética , Humanos , Soros Imunes , Metilação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Polimerase III/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Coelhos , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Relação Estrutura-Atividade , tRNA Metiltransferases/metabolismoRESUMO
U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end. The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing. Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24. Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix. These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle.
Assuntos
Splicing de RNA , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Fúngico/química , RNA Fúngico/genética , RNA Nuclear Pequeno/química , Proteínas de Ligação a RNA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Supressão GenéticaRESUMO
The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.
Assuntos
Proteínas Fúngicas/genética , Splicing de RNA/genética , RNA Nuclear Pequeno/genética , RNA/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Spliceossomos/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Fenótipo , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Fúngico/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Alinhamento de Sequência , Supressão Genética/genética , TemperaturaRESUMO
Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3'-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3'-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.
Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , Ribonucleoproteínas/biossíntese , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Transcrição GênicaRESUMO
We have fortuitously identified a nucleotide sequence that decreases expression of a reporter gene in the yeast Saccharomyces cerevisiae 20-fold when inserted into an intron. The primary effect of the insertion is a decrease in pre-mRNA abundance accompanied by the appearance of 3'-truncated transcripts, consistent with premature transcriptional termination and/or pre-mRNA degradation. Point mutations in the cis element relieve the negative effect, demonstrating its sequence specificity. A novel yeast protein, named Nrd1, and a previously identified putative helicase, Sen1, help mediate the negative effect of the cis element. Sen1 is an essential nuclear protein that has been implicated in a variety of nuclear functions. Nrd1 has hallmarks of a heterogeneous nuclear ribonucleoprotein, including an RNA recognition motif, a region rich in RE and RS dipeptides, and a proline- and glutamine-rich domain. An N-terminal domain of Nrd1 may facilitate direct interaction with RNA polymerase II. Disruption of the NRD1 gene is lethal, yet C-terminal truncations that delete the RNA recognition motif and abrogate the negative effect of the cis element nevertheless support cell growth. Thus, expression of a gene containing the cis element could be regulated through modulation of the activity of Nrd1. The recent identification of Nrd1-related proteins in mammalian cells suggests that this potential regulatory pathway is widespread among eukaryotes.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , DNA Helicases , Dados de Sequência Molecular , Mutação , RNA Helicases , Proteínas de Ligação a RNA , Alinhamento de SequênciaRESUMO
U6 RNA enters the spliceosome base paired with U4 RNA, but dissociates from U4 RNA before the catalytic steps of splicing. We have identified a cold-sensitive lethal mutation in U4 RNA (U4-cs1) that blocks the splicing pathway after U4/U6 complex formation, but before the first catalytic step of splicing. Remarkably, selection for suppressors of the cold-sensitive growth of the U4-cs1 strain yielded a tandem duplication of the highly conserved ACAGA sequence of U6 RNA (U6-Dup). The ACAGA sequence plays an essential role in spliceosome assembly and in the second catalytic step of pre-mRNA splicing; one or both of these roles involves direct base pairing to the pre-mRNA 5' splice site. In a U4-cs1/U6-Dup double-mutant strain grown at low temperature, the upstream ACAGA sequence of U6 RNA is required for suppression of the U4 mutation, whereas the downstream ACAGA sequence is required for other essential functions. Based on the sequence requirements for function of the upstream ACAGA element of U6-Dup, we propose that it pairs with the pre-mRNA 5' splice site during incorporation of the U4/U6 complex into the spliceosome and that the subsequent dissociation of U4 RNA exposes the downstream ACAGA sequence, which functions in the catalytic steps. The properties of this mutant U4/U6 complex provide compelling in vivo evidence that U6 RNA normally base pairs with the 5' splice site before disruption of its pairing with U4 RNA.
Assuntos
Família Multigênica , RNA Nuclear Pequeno , Spliceossomos/genética , Composição de Bases , Sequência de Bases , Divisão Celular/genética , Sequência Conservada , Genes Letais , Genes Supressores , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Splicing de RNA , RNA Fúngico , RNA Mensageiro , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element.
Assuntos
Genes Fúngicos , Genes Letais , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Transcrição Gênica , Alelos , Composição de Bases , Sequência de Bases , Sítios de Ligação , Sequência Consenso , DNA Fúngico/genética , DNA Fúngico/metabolismo , Expressão Gênica , Genes Recessivos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Homologia de Sequência do Ácido NucleicoRESUMO
Large-scale changes in RNA secondary structure, such as those that occur in some of the spliceosomal RNAs during pre-mRNA splicing, have been proposed to be catalyzed by ATP-dependent RNA helicases. Here we show that deproteinized human U4/U6 spliceosomal RNA complex, which has the potential for extensive intermolecular base pairing, contains a cis-acting element that promotes its dissociation into free U4 and U6 RNAs. The destabilzing element corresponds to the bae of putative intramolecular stem in U6 RNA that includes the 3' three-quarters of the molecule. Oligonucleotides expected to compete for U6 RNA 3' stem formation promote assembly of the human U4/U6 RNA complex under conditions that otherwise result in dissociation of the U4/U6 complex. Truncation of the putative 3' stem-forming sequences in U6 RNA by oligonucleotide-directed RNase H cleavage increases the melting temperature of the U4/U6 RNA complex by almost 20 degree C, to a level commensurate with its intermolecular base-pairing potential. We conclude that the stability of the competing human U6 RNA intramolecular 3' stem, combined with a low activation energy for conformational rearrangement, causes the human U4/U6 RNA complex to be intrinsically unstable despite its base-pairing potential. Therefore a helicase activity may not be necessary for disassembly of the human U4/U6 complex during activation of the spliceosome. We propose that a previously identified base-pairing interaction between U6 and U2 RNAs may stabilize the human U4/U6 RNA complex by antagonizing U6 RNA 3' stem formation.
Assuntos
RNA/química , Ribonucleoproteína Nuclear Pequena U4-U6/química , Spliceossomos , Sequência de Bases , Teste de Complementação Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos , Relação Estrutura-AtividadeRESUMO
The Saccharomyces cerevisiae U6 RNA gene (SNR6), which is transcribed by RNA polymerase III, has an unusual combination of promoter elements: an upstream TATA box, an intragenic A block, and a downstream B block. In tRNA genes, the A and B blocks are binding sites for the transcription initiation factor TFIIIC, which positions TFIIIB a fixed distance upstream of the A block. However, in vitro transcription of SNR6 with purified components requires neither TFIIIC nor the A and B blocks, presumably because TFIIIB recognizes the upstream sequences directly. Here we demonstrate that TFIIIB placement on SNR6 in vivo is directed primarily by the TFIIIC-binding elements rather than by upstream sequences. We show that the A block is a stronger start site determinant than the upstream sequences when the two are uncoupled by an insertion mutation. Furthermore, while TFIIIC-independent in vitro transcription of SNR6 is highly sensitive to TATA box point mutations, in vivo initiation on SNR6 is only marginally sensitive to such mutations unless the A block is mutated. Intriguingly, a deletion downstream of the U6 RNA coding region that reduces A-to-B block spacing also increases in vivo dependence on the TATA box. Moreover, this deletion results in the appearance of micrococcal nuclease-hypersensitive sites in the TFIIIB chromatin footprint, indicating that TFIIIB binding is disrupted by a mutation 150 bp distant. This and additional chromatin footprinting data suggest that SNR6 is assembled into a nucleoprotein complex that facilitates the TFIIIC-dependent binding of TFIIIB.
Assuntos
DNA Fúngico/metabolismo , Genes Fúngicos , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/metabolismo , TATA Box , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Primers do DNA , DNA Fúngico/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , RNA Fúngico/biossíntese , RNA Fúngico/genética , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae/genética , Deleção de Sequência , Relação Estrutura-Atividade , Fator de Transcrição TFIIB , Fatores de Transcrição/isolamento & purificaçãoRESUMO
U6 small nuclear RNA (snRNA) is an essential component of the spliceosome, the ribonucleoprotein complex that carries out the splicing of pre-mRNAs. The precise function of U6 RNA is unknown, but it has been proposed to participate directly in catalysis of the splicing reaction. We present biochemical and genetic evidence for an intramolecular stem/loop structure in the 3' half of U6 RNA of the yeast Saccharomyces cerevisiae that is mutually exclusive with the intermolecular base-pairing between U6 RNA and U4 snRNA. Strains with mutations that stabilize the U6 RNA 3'-intramolecular stem exhibit cold-sensitive growth and accumulate free U4 RNA, indicative of a block in U4/U6 snRNP assembly. The cold sensitivity can be partially suppressed by overexpression of U4 RNA. Mutations that disrupt base-pairing in the intramolecular 3' stem and mutations elsewhere in U6 RNA also suppress the growth defect. We conclude that a large conformational switch, involving melting of the U6 RNA 3' stem, is required for U4/U6 snRNP assembly. We hypothesize that formation of the U6 RNA intramolecular 3' stem after U4 RNA leaves the assembled spliceosome serves to activate U6 RNA for splicing by juxtaposing regions in U6 RNA that interact with U2 small nuclear RNA.
Assuntos
Conformação de Ácido Nucleico , Splicing de RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/química , Sequência de Bases , Dados de Sequência Molecular , Mutação , Fenótipo , RNA Fúngico/química , RNA Fúngico/genética , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Supressão GenéticaRESUMO
The promoters of vertebrate and yeast U6 small nuclear RNA genes are structurally dissimilar, although both are recognized by RNA polymerase III. Vertebrate U6 RNA genes have exclusively upstream promoters, while the U6 RNA gene from the yeast Saccharomyces cerevisiae (SNR6) has internal and downstream promoter elements that match the tRNA gene intragenic A- and B-block elements, respectively. Substitution of the SNR6 A or B block greatly diminished U6 RNA accumulation in vivo, and a subcellular extract competent for RNA polymerase III transcription generated nearly identical DNase I protection patterns over the SNR6 downstream B block and a tRNA gene intragenic B block. We conclude that the SNR6 promoter is functionally similar to tRNA gene promoters, although the effects of extragenic deletion mutations suggest that the downstream location of the SNR6 B block imposes unique positional constraints on its function. Both vertebrate and yeast U6 RNA genes have an upstream TATA box element not normally found in tRNA genes. Substitution of the SNR6 TATA box altered the site of transcription initiation in vivo, while substitution of sequences further upstream had no effect on SNR6 transcription. We present a model for the SNR6 transcription complex that explains these results in terms of their effects on the binding of transcription initiation factor TFIIIB.
Assuntos
Genes Fúngicos , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Alelos , Animais , Sequência de Bases , Sondas de DNA , Desoxirribonuclease I , Íntrons , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , RNA Fúngico/genética , Deleção de Sequência , TATA Box , Fator de Transcrição TFIIIB , Fatores de Transcrição/metabolismo , VertebradosRESUMO
Vertebrate genes coding for U6 small nuclear RNA are transcribed by RNA polymerase III (pol III), using only upstream promoter elements rather than the A and B block internal control regions typical of most pol III transcription units. We show that expression of the U6 gene from the yeast Saccharomyces cerevisiae has two unexpected features: it requires a B block promoter element, and this element is located in a novel position, 120 bp downstream of the coding region. In tRNA genes, the B block is the primary binding site for transcription factor (TF) IIIC, whose function is to promote the subsequent binding of TFIIIB. Both factors are thus implicated in yeast U6 gene transcription. We present a model of the U6 transcription complex based on the structure of yeast and vertebrate U6 promoters.
Assuntos
Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Northern Blotting , Clonagem Molecular , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Ribossômico/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , RNA Polimerase III/genética , RNA Ribossômico 5S/genética , Mapeamento por Restrição , Fatores de Transcrição/metabolismoRESUMO
Homologues of each of the five metazoan snRNAs required for pre-mRNA splicing have recently been identified in the budding yeast Saccharomyces cerevisiae on the basis of shared structural elements and evidence of similar roles during splicing. However, the spliceosomal snRNAs in this yeast are up to six times larger than their mammalian counterparts, suggesting that they may perform additional, perhaps species-specific, functions in the pre-mRNA processing pathway. We have undertaken a survey of 23 other budding yeasts to determine whether increased snRNA size is unique to Sacch. cerevisiae and, if not, to look for common structural motifs among homologous snRNAs. Our studies reveal that the spliceosomal snRNAs exhibit a surprising degree of size variation among these species. Furthermore, partial sequence analysis has identified a specific domain in the U6 snRNA which accounts for the observed size polymorphisms.