期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：46
页码：E7169-E7175
DOI：10.1073/pnas.1605873113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe splicing of human pre-mRNAs is tightly controlled and regulated during the assembly of the spliceosome onto pre-mRNA introns. Recognition of regulatory RNA sequence motifs by splicing factors is an essential early step during spliceosome assembly. We combine single-pair FRET and NMR to show that the recognition of the 3' splice site in pre-mRNA introns by the essential heterodimeric splicing factor U2 auxiliary factor (U2AF) involves conformational dynamics and population shifts of its RNA binding domains between open and closed conformations. Unexpectedly, the small subunit U2AF35 facilitates the recognition of weak splice sites by a population shift of the RNA binding domains of U2AF65 toward the open conformation. Notably, disease-linked mutations in U2AF65 do not affect RNA or U2AF35 binding. An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3' splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3' splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3' splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.
