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首页> 外文期刊>Frontiers in Molecular Biosciences >HMGA1a Induces Alternative Splicing of the Estrogen Receptor-αlpha Gene by Trapping U1 snRNP to an Upstream Pseudo-5′ Splice Site
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HMGA1a Induces Alternative Splicing of the Estrogen Receptor-αlpha Gene by Trapping U1 snRNP to an Upstream Pseudo-5′ Splice Site

机译：HMGA1a通过将U1 snRNP捕获到上游Pseudo-5'剪接位点诱导雌激素受体αlpha基因的选择性剪接

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Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells. Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2’-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a. RNA-binding of HMGA1a was checked by RNA-EMSA. In vitro splicing assay was performed to check the direct involvement of HMGA1a in splicing regulation. RNA-EMSA assay in the presence of purified U1 snRNP was performed with psoralen UV crosslinking to check complex formation of HMGA1a-U1 snRNP at the upstream pseudo-5’ splice site of exon 1. Results: HMGA1a induced exon skipping of a shortened exon 1 of ERα in in vitro splicing assays that was blocked by the HMGA1a RNA decoy and sequence-specific RNA-binding was confirmed by RNA-EMSA and ITC measurements. RNA-EMSA combined with psoralen UV crosslinking showed that HMGA1a trapped purified U1 snRNP at the upstream pseudo-5’ splice site. Conclusions: Regulation of ERα alternative splicing by an HMGA1a-trapped U1 snRNP complex at the upstream 5’ splice site of exon 1 offers novel insight on 5’ splice site regulation by U1 snRNP as well as a promising target in breast cancer therapy where alternative splicing of ERα is involved.
机译：目的：高迁移率的A组蛋白1a（HMGA1a）蛋白是与DNA结合的转录因子，但最近的研究表明，它通过RNA结合发挥了新的功能。提示我们破译HMGA1a诱导的雌激素受体α（ERα）选择性剪接的机制，最近我们报道该机制将改变tamoxifen对MCF-7 TAMR1细胞的敏感性。方法：通过瞬时表达HMGA1a和与HMGA1a结合的RNA诱饵（HMGA1a RNA结合位点的2'-O-甲基化RNA）进行瞬时表达，评估MCF-7乳腺癌细胞中全长ERα66及其异构体ERα46的内源表达。 。通过RNA-EMSA检查HMGA1a的RNA结合。进行体外剪接测定以检查HMGA1a是否直接参与剪接调控。在存在纯化的U1 snRNP的情况下，通过补骨脂素UV交联进行RNA-EMSA分析，以检查HMGA1a-U1 snRNP在外显子1的上游伪5'剪接位点处的复合物形成。结果：HMGA1a诱导外显子跳过缩短的外显子1。 RNA-EMSA和ITC测量证实了HMGA1a RNA诱捕剂阻断的ERα在体外剪接测定中的表达，并确认了序列特异性RNA结合。 RNA-EMSA与补骨脂素的UV交联表明，HMGA1a在上游伪5'剪接位点捕获了纯化的U1 snRNP。结论：HMGA1a捕获的U1 snRNP复合体在外显子1的上游5'剪接位点调控ERα选择性剪接提供了关于U1 snRNP调控5'剪接位点的新见解，也是乳腺癌治疗中有希望的替代靶点涉及ERα。

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