Voith von Voithenberg Lena, Sánchez-Rico Carolina, Kang Hyun-Seo, Madl Tobias, Zanier Katia, Barth Anders, Warner Lisa R, Sattler Michael, Lamb Don C
Department of Chemistry, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.
NanoSystems Initiative Munich, Ludwig-Maximilians University, Munich, D-81377 Munich, Germany.
Proc Natl Acad Sci U S A. 2016 Nov 15;113(46):E7169-E7175. doi: 10.1073/pnas.1605873113. Epub 2016 Oct 31.
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.
人类剪接体组装到前体mRNA上的一个重要早期步骤涉及U2辅助因子（U2AF）对3'剪接位点中调控性RNA顺式元件的识别。U2AF异源二聚体的大亚基（U2AF65）和小亚基（U2AF35）分别与多嘧啶序列（Py序列）和AG二核苷酸结合。U2AF65的串联RNA识别基序结构域（RRM1、2）在游离形式以及与真正的Py序列RNA配体结合时，会呈现封闭/无活性和开放/活性构象。为了研究U2AF65识别3'剪接位点的分子机制和动力学以及U2AF35在U2AF异源二聚体中的作用，我们结合了单对荧光共振能量转移（FRET）和核磁共振（NMR）实验。在没有RNA的情况下，RRM1、2结构域排列在亚毫秒时间尺度上具有高度动态性，在封闭和开放构象之间切换。添加结合亲和力（强度）不断增加的Py序列RNA配体逐渐使平衡向开放构象移动。值得注意的是，在存在强Py序列的情况下，蛋白质-RNA复合物是刚性的，但在存在弱Py序列时表现出内部运动。令人惊讶的是，U2AF35的UHM结构域与U2AF65的RRM1相互作用，在存在和不存在弱Py序列的情况下，都会增加U2AF65的RRM1、2开放排列的比例。这些数据表明，U2AF异源二聚体通过向U2AF65开放构象的动态群体转移来促进剪接体组装，以利于识别3'剪接位点处的弱Py序列。该异源二聚体的结构和RNA结合不受癌症相关的骨髓增生异常综合征突变体的影响。
