title: Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing.
abstract: SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35)  proteins. Interaction studies among U2AF(35)  paralogs and between U2AF(35)  and U1-70K revealed that U2AF(35)  can form homo- or heterodimers and that U2AF(35)  proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35)  and modulates AS.
authors.affiliation: Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Irene S
authors.initials: IS
authors.lastname: Day
authors.forename: Maxim
authors.initials: M
authors.lastname: Golovkin
authors.forename: Saiprasad G
authors.initials: SG
authors.lastname: Palusa
authors.forename: Alicia
authors.initials: A
authors.lastname: Link
authors.forename: Gul S
authors.initials: GS
authors.lastname: Ali
authors.forename: Julie
authors.initials: J
authors.lastname: Thomas
authors.forename: Dale N
authors.initials: DN
authors.lastname: Richardson
authors.forename: Anireddy S N
authors.initials: AS
authors.lastname: Reddy
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; C483303:SR45 protein, Arabidopsis; D000072668:Splicing Factor U2AF; C000591217:U1-70K protein, Arabidopsis; C000591218:U2AF(35)b protein, Arabidopsis; D000068103:Serine-Arginine Splicing Factors