RESUMO
The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5' splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5' splice site, and aberrant cleavage at the 5' splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5' splice site selection in the first splicing step.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Fatores de Processamento de RNA/genética , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Sequência de Bases/genética , Domínio Catalítico/genética , Humanos , Íntrons/genética , Mutação/genética , Sítios de Splice de RNA/genética , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Dedos de Zinco/genéticaRESUMO
Cwc24 is an essential splicing factor but only transiently associates with the spliceosome, with an unknown function. The protein contains a RING finger and a zinc finger domain in the carboxyl terminus. The human ortholog of Cwc24, RNF113A, has been associated with the disorder trichothiodystrophy. Here, we show that the zinc finger domain is essential for Cwc24 function, while the RING finger domain is dispensable. Cwc24 binds to the spliceosome after the Prp19-associated complex and is released upon Prp2 action. Cwc24 is not required for Prp2-mediated remodeling of the spliceosome, but the spliceosome becomes inactive if remodeling occurs before the addition of Cwc24. Cwc24 binds directly to pre-mRNA at the 5' splice site, spanning the splice junction. In the absence of Cwc24, U5 and U6 modes of interaction with the 5' splice site are altered, and splicing is very inefficient, with aberrant cleavage at the 5' splice site. Our data suggest roles for Cwc24 in orchestrating organization of the spliceosome into an active configuration prior to Prp2-mediated spliceosome remodeling and in promoting specific interaction of U5 and U6 with the 5' splice site for fidelity of 5' splice site selection.
Assuntos
Biocatálise , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA/metabolismo , Splicing de RNA/genética , Sequência de Bases , Reagentes de Ligações Cruzadas/metabolismo , Ligação Proteica , Domínios Proteicos , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction. The stable association of Cwc22 with the spliceosome requires the presence of the NTC but is independent of Prp2. Although Cwc22 is not required for the recruitment of Prp2 to the spliceosome, it is essential for the function of Prp2 in promoting the release of the U2 components SF3a and SF3b. In the absence of Cwc22, Prp2 can bind to the spliceosome but is dissociated upon ATP hydrolysis without promoting the release of SF3a/b. Thus, Cwc22 represents a novel ATP-dependent step one factor besides Prp2 and Spp2 and has a distinct role from that of Spp2 in mediating the function of Prp2.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA Fúngico/genética , Genes Fúngicos , Modelos Biológicos , Dados de Sequência Molecular , Splicing de RNA , Fatores de Processamento de RNA , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn(2+). These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5' splice site and the branch point during the first catalytic reaction.
Assuntos
Biocatálise , RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , Splicing de RNA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Biocatálise/efeitos dos fármacos , Temperatura Alta , Manganês/farmacologia , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , Fatores de Processamento de RNA , Saccharomyces cerevisiae/efeitos dos fármacos , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismoRESUMO
The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.
