School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR.
Department of Health Technology and Informatics The Hong Kong Polytechnic University Hong Kong SAR.
J Am Heart Assoc. 2020 Mar 17;9(6):e010240. doi: 10.1161/JAHA.118.010240. Epub 2020 Mar 11.
Background Pin2/TRF1-interacting protein, PinX1, was previously identified as a tumor suppressor. Here, we discovered a novel transcript variant of mPinX1 (mouse PinX1), mPinX1t (mouse PinX1t), in embryonic stem cells (ESCs). The aims of this investigation were (1) to detect the presence of mPinX1 and mPinX1t in ESCs and their differentiation derivatives; (2) to investigate the role of mPinX1 and mPinX1t on regulating the characteristics of undifferentiated ESCs and the cardiac differentiation of ESCs; (3) to elucidate the molecular mechanisms of how mPinX1 and mPinX1t regulate the cardiac differentiation of ESCs. Methods and Results By 5' rapid amplification of cDNA ends, 3' rapid amplification of cDNA ends, and polysome fractionation followed by reverse transcription-polymerase chain reaction, mPinX1t transcript was confirmed to be an intact mRNA that is actively translated. Western blot confirmed the existence of mPinX1t protein. Overexpression or knockdown of mPinX1 (both decreased mPinX1t expression) both decreased while overexpression of mPinX1t increased the cardiac differentiation of ESCs. Although both mPinX1 and mPinX1t proteins were found to bind to cardiac transcription factor mRNAs, only mPinX1t protein but not mPinX1 protein was found to bind to nucleoporin 133 protein, a nuclear pore complex component. In addition, mPinX1t-containing cells were found to have a higher cytosol-to-nucleus ratio of cardiac transcription factor mRNAs when compared with that in the control cells. Our data suggested that mPinX1t may positively regulate cardiac differentiation by enhancing export of cardiac transcription factor mRNAs through interacting with nucleoporin 133. Conclusions We discovered a novel transcript variant of mPinX1, the mPinX1t, which positively regulates the cardiac differentiation of ESCs.
背景 Pin2/TRF1 相互作用蛋白 PinX1 先前被鉴定为一种肿瘤抑制因子。在这里，我们在胚胎干细胞 (ESC) 中发现了 mPinX1 (小鼠 PinX1) 的一种新的转录变体 mPinX1t (小鼠 PinX1t)。本研究的目的是：(1) 检测 mPinX1 和 mPinX1t 在 ESC 及其分化衍生物中的存在；(2) 研究 mPinX1 和 mPinX1t 对调节未分化 ESC 特征和 ESC 心脏分化的作用；(3) 阐明 mPinX1 和 mPinX1t 调节 ESC 心脏分化的分子机制。方法和结果通过 5' 快速扩增 cDNA 末端、3' 快速扩增 cDNA 末端和多核糖体分级分离，然后进行逆转录-聚合酶链反应，证实 mPinX1t 转录本是一种活跃翻译的完整 mRNA。Western blot 证实了 mPinX1t 蛋白的存在。mPinX1 的过表达或敲低 (均降低了 mPinX1t 的表达) 均降低了 ESC 的心脏分化，而过表达 mPinX1t 则增加了 ESC 的心脏分化。尽管 mPinX1 和 mPinX1t 蛋白都被发现与心脏转录因子 mRNAs 结合，但只有 mPinX1t 蛋白而不是 mPinX1 蛋白被发现与核孔复合体成分核孔蛋白 133 结合。此外，与对照细胞相比，含有 mPinX1t 的细胞中心脏转录因子 mRNAs 的细胞质到细胞核比值更高。我们的数据表明，mPinX1t 可能通过与核孔蛋白 133 相互作用增强心脏转录因子 mRNAs 的输出，从而正向调节 ESC 的心脏分化。结论我们发现了 mPinX1 的一种新的转录变体 mPinX1t，它正向调节 ESC 的心脏分化。
