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Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing

Publication Type:Journal Article
Year of Publication:2012
Authors:Day, IS, Golovkin, M, Palusa, SG, Link, A, Ali, GS, Thomas, J, Richardson, DN, Reddy, ASN
Journal:The Plant Journal
Volume:71
Issue:6
Date Published:2012
ISBN Number:1365-313X
Keywords:alternative splicing, Arabidopsis, pre-mRNA splicing, serine/arginine-rich protein, splicing regulator, SR30, SR45
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 U2AF35b 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 (U2AF35). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF35. Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF35 proteins. Interaction studies among U2AF35 paralogs and between U2AF35 and U1-70K revealed that U2AF35 can form homo- or heterodimers and that U2AF35 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 U2AF35b 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 U2AF35b. These results suggest that SR45 recruits U1snRNP and U2AF to 5′ and 3′ splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF35 and modulates AS.

URL:http://dx.doi.org/10.1111/j.1365-313X.2012.05042.x
Short Title:The Plant Journal
Taxonomic name: 
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