Abstract
The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.
Highlights
40) consists of a pair of WW domains followed by several FF domains
The Prp40 WW domains bind to proline-rich sequences in the branch point-binding protein and the U5 snRNP-associated protein Prp8 [2, 3], the region spanning the FF domains has been shown to associate with the 5Ј end of U1 snRNP [4] and to interact with at least two different proteins as follows: the splicing factor Clf1/Syf3p [5,6,7], and the C-terminal domain (CTD) of the largest subdomain of RNA polymerase II (RNAPII) [8]
Proteins annotated in data bases as hypothetical were classified by domain architecture and length of the linkers connecting the WW domains as follows: proteins containing two WW domains separated by a short (ϳ15 aa) conserved linker were initially classified as similar to FBP11 proteins, whereas proteins with two or more WW domains separated by long, variable linkers were categorized as CA150-related
Summary
40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5 end of U1 small nuclear RNA and to interact directly with two proteins, the Clf (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). The Prp WW domains bind to proline-rich sequences in the branch point-binding protein and the U5 snRNP-associated protein Prp8 [2, 3], the region spanning the FF domains has been shown to associate with the 5Ј end of U1 snRNP [4] and to interact with at least two different proteins as follows: the splicing factor Clf1/Syf3p (crooked neck-like factor 1/synthetic lethal with cdc 40) [5,6,7], and the C-terminal domain (CTD) of the largest subdomain of RNA polymerase II (RNAPII) [8]. The diversity in the function and in the structure of these Prp FF domain binding partners raises important questions as to whether distinct binding specificities exist in FF domains and how the FF domain fold enables this potential promiscuity in ligand binding
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
