Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation.
The active 17S U2 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 SF3b and SF3a with the 12S U2 snRNP. We have analyzed the function of individual subunits of human SF3a (SF3a60, SF3a66, and SF3a120) by testing recombinant proteins, expressed in insect cells, in various in vitro assays. The recombinant subunits readily form the SF3a heterotrimer, where SF3a60 and SF3a66 interact with SF3a120, but not with each other. All SF3a subunits are essential for the formation of the mature 17S U2 snRNP and the prespliceosome. Single subunits engage in interactions with the 15S U2 snRNP (consisting of the 12S U2 snRNP and SF3b), and SF3a60 appears to play a major role in recruiting SF3a120 into the U2 particle. Analysis of functional domains in SF3a60 and SF3a66 identified interaction sites for SF3a120 in their N-terminal portions. C(2)H(2)-type zinc finger domains mediate the integration of SF3a60 and SF3a66 into the U2 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 U2 snRNP. Finally, we demonstrate that all domains required for interactions within the SF3a heterotrimer and the formation of the 17S U2 snRNP are also necessary to assemble the prespliceosome.
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Département de Biologie Cellulaire, Université de Genève, CH-1211 Geneva 4, Switzerland.
Krämer
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D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics; D014162:Transfection; D016335:Zinc Fingers
D021122:Protein Subunits; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; C088049:splicing factor 3a