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

hnRNP A2/B1 对胰岛素分泌颗粒蛋白产生的调节

• 批准号: 455353954
• 负责人: Professor Michele Solimena
• 金额: --
• 依托单位: Paul-Langerhans-Institut Dresden (PLID) des Helmholtz-Zentrums München am Universitätsklinikum Carl Gustav Carus der TU Dresden
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/455353954?language=en
• 关键词: regulation; insulin; secretory; granule; protein

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.

mrna与rna结合蛋白（rbp）转录后动态组装成核糖核蛋白复合物（RNPs）微调基因表达。具体来说，rbp调节每个mRNA的剪接、输出、储存、翻译和降解，包括其靶向多体、内质网或细胞质RNA颗粒。此外，rbp与功能相关mrna之间共享的基序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节与血糖相关的胰岛素产生。高血糖迅速促进胰岛素和其他胰岛素分泌颗粒货物的生物合成，如PC1/3、PC2和ICA512，而不影响相应的mrna水平。我们发现，在静止的MIN6细胞中，一组共同的rbp结合了胰岛素1、胰岛素2、剪接的胰岛素2、PC2和ICA512 mrna的5'- utr，而另一组共享的rbp在葡萄糖刺激下协调这些mrna的翻译。在所有测试的胰岛素分泌颗粒货物mrna中，一个新的RBP是hnRNP A2/B1。我们在Insulin1 mRNA的5'-UTR中绘制了hnRNP A2/B1结合位点，并检测到hnRNP A2/B1结合位点在突变后减少。此外，与WT细胞相比，Hnrnpa2b1 /- MIN6细胞显示胰岛素1 mRNA和总胰岛素蛋白水平降低。最后，我们发现在静息培养的MIN6细胞和人β细胞中，hnRNP A2/B1在细胞质RNA颗粒中富集。在此，我们建议进一步开展这些研究，以更好地了解β细胞的生理和病理生理。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mrna的动态表达。接下来，我们将使用带有RNA适体和荧光rbp标记的RNA，通过FLIM-FRET成像研究RNP动力学，以空间和时间分辨的方式破译胰岛素颗粒蛋白mrna的RNP编码。最后，我们将研究hnRNP A2/B1在人类β细胞中的作用，以及hnRNP A2/B1阳性RNA颗粒的存在是否与糖尿病中的β细胞功能障碍相关。