
<ArticleTitle>Domains in human splicing factors <prot>SF3a60</prot> and <prot>SF3a66</prot> required for binding to <prot>SF3a120</prot>, assembly of the 17S <prot>U2</prot> snRNP, and prespliceosome formation.</ArticleTitle>
<AbstractText>The active 17S <prot>U2</prot> small nuclear ribonucleoprotein particle (snRNP), which binds to the intron branch site during the formation of the prespliceosome, is assembled in vitro by sequential interactions of the essential splicing factors <prot>SF3b</prot> and <prot>SF3a</prot> with the 12S <prot>U2</prot> snRNP. We have analyzed the function of individual subunits of human <prot>SF3a</prot> (<prot>SF3a60</prot>, <prot>SF3a66</prot>, and <prot>SF3a120</prot>) by testing recombinant proteins, expressed in insect cells, in various in vitro assays. The recombinant subunits readily form the <prot>SF3a</prot> heterotrimer, where <prot>SF3a60</prot> and <prot>SF3a66</prot> interact with <prot>SF3a120</prot>, but not with each other. All <prot>SF3a</prot> subunits are essential for the formation of the mature 17S <prot>U2</prot> snRNP and the prespliceosome. Single subunits engage in interactions with the 15S <prot>U2</prot> snRNP (consisting of the 12S <prot>U2</prot> snRNP and <prot>SF3b</prot>), and <prot>SF3a60</prot> appears to play a major role in recruiting <prot>SF3a120</prot> into the <prot>U2</prot> particle. Analysis of functional domains in <prot>SF3a60</prot> and <prot>SF3a66</prot> identified interaction sites for <prot>SF3a120</prot> in their N-terminal portions. C(2)H(2)-type zinc finger domains mediate the integration of <prot>SF3a60</prot> and <prot>SF3a66</prot> into the <prot>U2</prot> snRNP, and we propose a model in which protein-protein interactions between the zinc finger domains and the Sm proteins, common to all spliceosomal snRNPs, contribute to the assembly of the 17S <prot>U2</prot> snRNP. Finally, we demonstrate that all domains required for interactions within the <prot>SF3a</prot> heterotrimer and the formation of the 17S <prot>U2</prot> snRNP are also necessary to assemble the prespliceosome.</AbstractText>
