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Molecular and Cellular Biology, October 2001, p. 6406-6417, Vol. 21, No. 19
Département de Biologie Cellulaire,
Université de Genève, CH-1211 Geneva 4, Switzerland
Received 23 April 2001/Returned for modification 28 May
2001/Accepted 6 July 2001
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. C2H2-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.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.19.6406-6417.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Domains in Human Splicing Factors SF3a60 and SF3a66 Required for
Binding to SF3a120, Assembly of the 17S U2 snRNP, and
Prespliceosome Formation
and
*
Corresponding author. Mailing address:
Département de Biologie Cellulaire, Sciences III,
Université de Genève, 30, quai Ernest-Ansermet, CH-1211
Geneva 4, Switzerland. Phone: 41-22-702-6750. Fax: 41-22-702-6750. E-mail: angela.kraemer{at}cellbio.unige.ch.
Present address: Immunology Division, Walter and Eliza Hall
Institute of Medical Research, The Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia.
Molecular and Cellular Biology, October 2001, p. 6406-6417, Vol. 21, No. 19
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.19.6406-6417.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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