期刊
RNA
卷 22, 期 2, 页码 265-277出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.054296.115
关键词
pre-mRNA processing factor 38; pre-mRNA splicing; protein-protein interactions; spliceosomal B complex; spliceosome; yeast two-hybrid analysis
资金
- Helmholtz Zentrum Berlin fur Materialien and Energie
- Freie Universitat Berlin
- Humboldt-Unversitat zu Berlin
- Max-Delbruck Centrum
- Leibniz-Institut fur Molekulare Pharmakologie
- Max-Planck Society
- Deutsche Forschungsgemeinschaft [WA1126/7-1, LU294/15-1]
Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel alpha-helices and lacks similarities to RNA binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage. as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein protein interaction platform that might organize the relative positioning of other proteins during splicing.
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