Laboratory of Gene Therapy, Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland.
Center for NeuroGenetics and the Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, 2033 Mowry Road, Gainesville, FL 32610, USA.
Nucleic Acids Res. 2018 Sep 28;46(17):9119-9133. doi: 10.1093/nar/gky565.
Muscleblind-like (MBNL) proteins are conserved RNA-binding factors involved in alternative splicing (AS) regulation during development. While AS is controlled by distribution of MBNL paralogs and isoforms, the affinity of these proteins for specific RNA-binding regions and their location within transcripts, it is currently unclear how RNA structure impacts MBNL-mediated AS regulation. Here, we defined the RNA structural determinants affecting MBNL-dependent AS activity using both cellular and biochemical assays. While enhanced inclusion of MBNL-regulated alternative exons is controlled by the arrangement and number of MBNL binding sites within unstructured RNA, when these sites are embedded in a RNA hairpin MBNL binds preferentially to one side of stem region. Surprisingly, binding of MBNL proteins to RNA targets did not entirely correlate with AS efficiency. Moreover, comparison of MBNL proteins revealed structure-dependent competitive behavior between the paralogs. Our results showed that the structure of targeted RNAs is a prevalent component of the mechanism of alternative splicing regulation by MBNLs.
肌萎缩侧索硬化症（MBNL）蛋白是保守的 RNA 结合因子，参与发育过程中的可变剪接（AS）调控。虽然 AS 受 MBNL 同源物和同工型的分布控制，以及这些蛋白质对特定 RNA 结合区域的亲和力及其在转录本中的位置，但目前尚不清楚 RNA 结构如何影响 MBNL 介导的 AS 调控。在这里，我们使用细胞和生化测定法来定义影响 MBNL 依赖性 AS 活性的 RNA 结构决定因素。虽然 MBNL 调节的可变外显子的增强包含受无结构 RNA 内 MBNL 结合位点的排列和数量控制，但当这些位点嵌入 RNA 发夹时，MBNL 优先结合到茎区的一侧。令人惊讶的是，MBNL 蛋白与 RNA 靶标的结合与 AS 效率不完全相关。此外，对 MBNL 蛋白的比较表明，在同系物之间存在结构依赖性竞争行为。我们的结果表明，靶向 RNA 的结构是 MBNLs 调节可变剪接的机制中的一个普遍组成部分。
