Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Nucleic Acids Res. 2020 Jun 4;48(10):5695-5709. doi: 10.1093/nar/gkaa293.
An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3' splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.
U2AF1 和 U2AF2 这两个剪接因子的异源二聚体在富含嘧啶（Py）的信号处起始拼接体组装，该信号位于主要的 3' 剪接位点之前。在骨髓增生异常综合征（MDS）患者中，U2AF1 常获得 S34F 编码突变。然而，U2AF1 亚基及其 S34F 突变对 U2AF2 构象的影响尚不清楚。在这里，我们利用单分子Förster 共振能量转移（FRET）来确定野生型或 S34F 取代的 U2AF1 对 U2AF2 及其剪接位点 RNA 复合物构象动力学的影响。在没有 RNA 的情况下，U2AF1 亚基稳定了一个高 FRET 值，通过结构引导的突变，该值对应于串联 U2AF2 RNA 识别基序（RRM）的封闭构象。当 U2AF 异源二聚体与强的、富含尿嘧啶的剪接位点结合时，U2AF2 切换到一个较低的 FRET 值，这一特征是 RRMs 侧对侧开放排列的结果。值得注意的是，U2AF 异源二聚体结合弱的、富含尿嘧啶的 Py 片段作为封闭和开放 U2AF2 构象的混合物，这一混合物由 S34F 突变来调节。开放和封闭的 U2AF2 之间的转变可能是 U2AF1 依赖性的退化 Py 片段剪接的基础，并有助于 MDS 患者中 S34F 失调剪接事件的一部分。
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