Protein-Protein-Interaktionen der U1snRNP-Komponente Prp40 und deren FF-Domänen in Saccharomyces cerevisiae

Abstract

The FF domain is conserved in all eukaryotes indicating an important biological role. Many FF domain proteins play a role in RNA metabolism and their FF domains typically occur in tandem arrays. The yeast splicing factor Prp40 an essential component of the U1snRNP contains four FF domains and appears to play a role in stabilization of the yeast commitment complex during spliceosome assembly. The function of the FF domains in this process is unclear. FF domains of Prp40 and the human proteins CA150 and HYPA/FBP11 are known to interact with the phosphorylated C-terminal domain (CTD) of the RNA polymerase II. By yeast genome-wide two-hybrid screens I found that FF domains of Prp40 specifically interact with Snu71 and Luc7, known components of the U1snRNP. Mapping experiments showed that the FF1 domain of Prp40 interacts with a C-terminal region within Luc7, also confirmed by in vitro binding assays, whereas FF2-3 domains interact with a C-terminal region within Snu71. Peptide array results showed an interaction region within Snu71 (NDVHY) with no similarity to the consensus motif for Luc7 (Φ[FHL]x(KR]x[GHL]) identified via substitution analysis. Mutations of involved amino acids within the Luc7 sequence showed not the expected effect in Y2H experiments with FF domains. Only a mutation of the lysine resulted in the loss of binding with Prp40, but only in slow growth of yeast cells with FF1. Also a peptide containing the identified interacting region from Luc7 did not block the binding of FF1 and Luc7 in competition assay. Comparison of the identified motif for Luc7 with previous characterised consensus sequences from FF domains revealed no similarity, indicating the identification of a novel binding site for FF-domains. To get more insight into the yeast U1snRNP interactions also all associated proteins were tested in Y2H experiments. But only the interaction from Prp40 with Snu71 and Luc7 was detected again, concluding that this complex is hold together also through protein-RNA interaction. In vivo FF-domain deletions in Prp40 produced a mutant with growth defect lacking FF domains four and three, probably due to a defect in splicing. This was tested by a functional splice test with two representative intron containing genes, with no influence on splicing. To achieve all intron containing genes in yeast also microarray-splice experiments were initiated to obtain genome wide results. Based on all interaction studies, we conclude, that these three U1snRNP proteins Prp40, Snu71 and Luc7 build a sub complex within the U1snRNP via FF-domain interactions, like U1-A, U1-70K and U1-C in the human U1snRNP.