HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/-KTS isoforms.
HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/-KTS isoforms.
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HuR 拮抗富含内含子嘧啶的序列在调节 WT1 /-KTS 亚型中的作用。
DOI：
10.1080/15476286.2015.1102831
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Lei Haixin
中科院分区：
文献类型：
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作者：
Li Hui;Hou Shuai;Hao Tian;Azam Sik;ar;Liu Caigang;Shi Lei;Lei Haixin
−+Kts和WT1+Kts亚型在蛋白质序列上只有3个氨基酸的差异，但在功能上存在显著差异。+/−亚型是由两个相邻的5‘端剪接位点在RNA水平上交替使用而产生的，然而，这两种亚型是如何被调控的，目前还不清楚。在这里，我们报告了对+/−kts亚型比率至关重要的富含内含子嘧啶的序列的鉴定，该序列的缺失或部分替换导致了完全/显著的向-kts亚型的转变。为了确定可以通过与元件结合来调节+/RNPKTS异构体的反式因子，我们使用体外转录的−进行了组装，其中有或没有富含嘧啶的序列。对纯化的RNPs进行了质谱分析，结果表明该元件与多种剪接因子有关。将这些因子与WT1报告基因共转染，发现HUR在报告基因水平上促进了−KTS亚型的产生。RNA免疫沉淀实验表明，HUR与WT1内含子9中的富含嘧啶的元件相互作用。进一步的证据表明，HUR的瞬时或稳定过表达导致内源−KTS亚型表达增强。此外，在293T、SW620、SNU387和AGS四种细胞系中，HUR基因被敲除后，内源性−KTS亚型的表达均降低。综上所述，这些数据表明，HUR与富含嘧啶的序列结合，并拮抗其调节WT1+/−KTS亚型的作用。
WT1 + KTS and −KTS isoforms only differ in 3 amino acids in protein sequence but show significant functional difference. The +/−KTS isoforms were generated by alternative usage of 2 adjacent 5’ splice sites at RNA level, however, how these 2 isoforms are regulated is still elusive. Here we report the identification of an intronic pyrimidine-rich sequence that is critical for the ratio of +/−KTS isoforms, deletion or partial replacement of the sequence led to full/significant shift to -KTS isoform. To identify trans-factors that can regulate +/−KTS isoforms via the binding to the element, we performed RNP assembly using in vitro transcribed RNA with or without the pyrimidine-rich sequence. Mass spectrometry analysis of purified RNPs showed that the element associated with many splicing factors. Co-transfection of these factors with WT1 reporter revealed that HuR promoted the production of −KTS isoform at the reporter level. RNA immuno-precipitation experiment indicated that HuR interacted with the pyrimidine-rich element in WT1 intron 9. We further presented evidence that transient or stable over-expression of HuR led to enhanced expression of endogenous −KTS isoform. Moreover, knockdown of HuR resulted in decreased expression of endogenous −KTS isoform in 293T, SW620, SNU-387 and AGS cell lines. Together, these data indicate that HuR binds to the pyrimidine-rich sequence and antagonize its effect in regulating WT1 +/−KTS isoforms.