Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, OR 97401, USA.
Nucleic Acids Res. 2010 Apr;38(7):2467-84. doi: 10.1093/nar/gkp1209. Epub 2010 Jan 13.
Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1's role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs.
肌肉萎缩症相关蛋白 1（MBNL1）调控可变剪接，是肌强直性营养不良症（DM）发病机制中的关键因子。在 DM 中，MBNL1 与扩展的 CUG/CCUG 重复 RNA 结合，导致剪接缺陷，从而引发疾病症状。为了了解 MBNL1 在 DM 疾病机制和可变剪接调控中的作用，我们试图鉴定其 RNA 结合基序。在已知的 MBNL1 结合位点上进行了掺杂 SELEX 实验。经过五轮 SELEX，MBNL1 选择富含嘧啶的 RNA，其中包含 YGCY 基序。将多个 YGCY 基序插入通常不依赖于 MBNL1 的剪接报告子中，足以促进 MBNL1 的调控。MBNL1 还通过 YGCY 基序调控 DM 中错剪接的 ATP2A1 前体 mRNA 的外显子 22 的剪接。在 24 种在 DM1 患者中发生异常剪接的前体 mRNA 转录本中搜索 YGCY 基序，相对于受调控的外显子，发现了一个有趣的模式。与 DM1 相比，在正常组织中排除的外显子上游的内含子区域富含 YGCY 基序。而在正常组织中包含的外显子下游的内含子区域富含 YGCY 基序。
