The regulation of insulin secretory granule protein production by hnRNP A2/B1

转录后动态组装mRNAs与RNA结合蛋白(RBPs)形成核糖核蛋白复合体(RNPs)微调基因表达。具体地说，限制性商业惯例调节每个单独的mRNA的剪接、输出、储存、翻译和降解，包括其靶向多聚体、内质网或细胞质RNA颗粒。此外，限制性商业惯例与功能相关的mRNAs共享的基序的结合允许后者的协调表达。胰岛β细胞利用转录后机制来快速调整它们相对于血糖的胰岛素产生。高血糖迅速促进胰岛素和其他胰岛素分泌颗粒的生物合成，如PC1/3、PC2和ICA512，而不会最初影响相应的mRNAs水平。我们发现，在静息的MIN6细胞中，一组共同的RBPs结合了Insulin1、Insulin2、剪接的Insulin2、PC2和ICA512mRNAs的5‘-UTRs，而另一组共享的RBPs协调了这些mRNAs在葡萄糖刺激下的翻译。在所有被测试的胰岛素分泌颗粒运输的mRNAs中共享一个新的RBP，即hnRNPA2/B1。我们将hnRNP A2/B1结合位点定位在Insulin1mRNA的5‘-UTR区，并检测到突变后hnRNP A2/B1结合减少。此外，与WT细胞相比，hnRNPA2B1/-MIN6细胞的胰岛素1mRNA和总胰岛素蛋白水平降低。最后，我们发现在培养的静息MIN6细胞和人β细胞中，hnRNP A2/B1富含细胞质RNA颗粒。在这里，我们建议进一步发展这些研究，以更好地了解β细胞的生理学和病理生理学。第一个目的是阐明hnRNP A2/B1如何控制胰岛素和其他分泌颗粒蛋白的mRNAs的动态表达。接下来，我们将使用标记有RNA适配子和荧光限制性商业惯例的RNA，用胶片FRET成像来研究RNP的动力学，以便以空间和时间分辨的方式破译胰岛素颗粒蛋白mRNAs的RNP编码。最后，我们将研究hnRNPA2/B1‘S在人β细胞中的作用，以及hnRNPA2/B1阳性RNA颗粒的存在是否与糖尿病的β细胞功能障碍有关。

Post-transcriptional dynamic assembly of mRNAs with RNA-binding proteins (RBPs) into ribonucleoprotein complexes (RNPs) fine-tunes gene expression. Specifically, RBPs regulate the splicing, export, storage, translation and degradation of each individual mRNA, including its targeting to polysomes, the endoplasmic reticulum or cytoplasmic RNA granules. Moreover, binding of RBPs to motifs shared among functionally related mRNAs allows for the coordinated expression of the latter. Pancreatic islet beta cells exploit post-transcriptional mechanisms to quickly adjust their insulin production relative to glycaemia. Hyperglycaemia rapidly enhances the biosynthesis of insulin and other insulin secretory granule cargoes, e.g. PC1/3, PC2 and ICA512, without initially affecting the corresponding mRNAs levels. We discovered that in resting MIN6 cells a common set of RBPs binds the 5'-UTRs of Insulin1, Insulin2, spliced Insulin2, PC2 and ICA512 mRNAs, while another set of shared RBPs coordinate the translation of these mRNAs upon glucose stimulation. A novel RBP shared among all tested mRNAs for insulin secretory granule cargoes is hnRNP A2/B1. We mapped hnRNP A2/B1-binding sites in the 5'-UTR of Insulin1 mRNA and detected reduced hnRNP A2/B1 binding upon their mutation. Furthermore, Hnrnpa2b1 /- MIN6 cells displayed reduced Insulin1 mRNA and total insulin protein levels compared to WT cells. Finally, we found that in resting MIN6 cells in culture and in human beta cells in situ hnRNP A2/B1 is enriched in cytoplasmic RNA granules. Here we propose to develop further these studies to better understand the physiology and pathophysiology of beta cells. The first aim is to elucidate how hnRNP A2/B1 controls the dynamic expression of mRNAs for insulin and other secretory granule proteins. Next, we shall study RNP dynamics with FLIM-FRET imaging using RNAs tagged with RNA aptamers and fluorescent RBPs in order to decipher the RNP code of mRNAs for insulin granule proteins in a space- and time-resolved manner. Finally, we shall investigate hnRNP A2/B1’s role in human beta cells and if the presence of hnRNP A2/B1 positive RNA granules correlates with beta cell dysfunction in diabetes.