Alternative splicing is one of the mechanisms how to regulate gene expression. Under different conditions, different mRNAs encoding proteins with different function, localization or stability can be made from one cellular transcript. The human hSlu7 protein affects the alternative splicing of some genes through alternative 3'splice site (3'SS) selection. Although it was thought that alternative splicing is absent from Saccharomyces cerevisiae, recent results argue against such conclusion. We therefore decided to characterize the function of the yeast Slu7 protein, which participates in the second step of splicing and is closely associated with the 3'SS selection. We focused on a highly conserved uncharacterized motif in the essential part of the Slu7 protein named the RED motif. Mutations in this motif caused second step splicing defects with some substrates and altered the alternative 3'SS usage ratio of some splicing constructs. Our results implicate a role for the RED motif in selecting proper 3'splice sites, especially the distal ones. Genetic interactions of slu7 mutations with PRP22 and PRP45 mutant alelles add to the intricate interaction network of splicing factors and suggest a possible role of Slu7p in facilitating the Prp22p association with the spliceosome.

The informational content of 3' splice sites is low and the mechanisms whereby they are selected are not clear. Here we enunciate a set of rules that govern their selection. For many introns, secondary structures are a key factor, because they occlude alternative 3'ss from the spliceosome and reduce the effective distance between the BS and the 3'ss to a maximum of 45 nucleotides. Further alternative 3'ss are disregarded by the spliceosome because they lie at 9 nucleotides or less from the branch site, or because they are weak splice sites. With these rules, we are able to explain the splicing pattern of the vast majority of introns in Saccharomyces cerevisiae. When in excess, L30 blocks the splicing of its own transcript by interfering with a critical rearrangement that is required for the proper recognition of the intron 3' end, and thus for splicing to proceed. We show that the protein Cbp80 has a role in promoting this rearrangement and therefore antagonizes splicing regulation by L30. / Tanto la información que define el sitio de splicing 3' como los mecanismos de selección del mismo son poco conocidos. En este trabajo, proponemos una serie de reglas que gobiernan esta selección. Las estructuras secundarias son claves en el caso de muchos intrones, porque son capaces de ocultar sitios de splicing alternativos 3' al spliceosoma, y además reducen la distancia efectiva entre el punto de ramificación y el sitio de splicing 3' a un máximo de 45 nucleotidos. Otros sitios de splicing alternativo 3' no son considerados por el spliceosoma como tales porque se encuentran a 9 nucleotidos o menos del punto de ramificación, o porque son sitios de splicing débiles. Con estas reglas somos capaces de explicar el splicing de la mayoría de intrones de Saccharomyces cerevisiae. El exceso de proteína L30 bloquea el splicing de su propio tránscrito porque interfiere con la reorganización necesaria para el correcto reconocimiento del 3' final del intrón, y por tanto de su splicing. Demostramos que la proteína Cbp80 está implicada en promover esta reorganización y que por tanto antagoniza la regulación del splicing por L30.