Loerch Sarah, Maucuer Alexandre, Manceau Valérie, Green Michael R, Kielkopf Clara L
From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and.
the Howard Hughes Medical Institute and Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
J Biol Chem. 2014 Jun 20;289(25):17325-37. doi: 10.1074/jbc.M114.558825. Epub 2014 May 2.
U2AF homology motifs (UHMs) mediate protein-protein interactions with U2AF ligand motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERα regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERα UHM is unknown. Here we present structures of the CAPERα UHM bound to a representative SF3b155 ULM at 1.7 Å resolution and, for comparison, in the absence of ligand at 2.2 Å resolution. The prototypical UHM/ULM interactions authenticate CAPERα as a bona fide member of the UHM family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERα in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERα UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERα molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously identified, UHM/ULM-mediated complexes of U2AF(65) and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.
U2AF同源基序（UHMs）介导与前体mRNA剪接因子的U2AF配体基序（ULMs）的蛋白质-蛋白质相互作用。含UHM的可变剪接因子Caperα调节促肿瘤血管内皮生长因子（VEGF）异构体的剪接，但Caperα UHM的分子靶点尚不清楚。在此，我们展示了Caperα UHM与代表性的SF3b155 ULM结合时分辨率为1.7 Å的结构，作为比较，还展示了其在无配体时分辨率为2.2 Å的结构。典型的UHM/ULM相互作用证实Caperα是UHM家族蛋白质的真正成员。我们在人细胞提取物中确定SF3b155是全长Caperα的相关含ULM的伙伴。对纯化的Caperα UHM与已知含ULM蛋白质结合的等温滴定量热法比较表明，高亲和力相互作用取决于完整的、内在无序的含有七个ULM样基序的SF3b155结构域的存在。结合的Caperα分子之间的相互作用导致在含SF3b155 ULM的结构域中出现两个高亲和力位点。结合先前鉴定的U2AF（65）和SPF45与SF3b155的UHM/ULM介导的复合物，这项工作证明了SF3b155为协调招募含UHM的剪接因子提供平台的能力。
