RESUMO
The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA, yielding mature message. Schizosaccharomyces pombe Cwf10 (homolog of Saccharomyces cerevisiae Snu114 and human U5-116K), an integral member of the U5 snRNP, is a GTPase that has multiple roles within the splicing cycle. Cwf10/Snu114 family members are highly homologous to eukaryotic translation elongation factor EF2, and they contain a conserved N-terminal extension (NTE) to the EF2-like portion, predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation and second-step catalysis. Characterization of Cwf10-NTE by various biophysical techniques shows that in solution the NTE contains regions of both structure and disorder. The first 23 highly conserved amino acids of the NTE are essential for its role in splicing but when overexpressed are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-ΔNTE cells. When the entire NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of independently incorporating into the spliceosome and improving splicing function, possibly indicating a role for the NTE in stabilizing conformational rearrangements during a splice cycle.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , GTP Fosfo-Hidrolases/genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Spliceossomos/metabolismoRESUMO
Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVß1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.