Intranuclear trafficking and export of single, native mRNA molecules analyzed by highresolution light sheet microscopy

信使核糖核蛋白颗粒（mRNPs）的输出可以被调节。这种调控可能对基因表达结果有很强的影响，但人们对其了解甚少。我们的目的是在昆虫模型系统中研究mRNP的贩运和出口机制。我们最近已经证明，我们可以使单一的，特异性的，天然的mRNP -在触手手蛾幼虫的唾液腺细胞中的Balbiani环（BR） 2 mRNP -可见，并跟踪它们在活细胞核质中的运动。我们现在计划在这个三方项目中使用这个系统。(i)应开发一种特殊的显微镜，具有彩色校正光片照明，以前所未有的灵敏度进行单分子观察；（ii）应检查在核质无染色质区域检测到的BR mRNP的不连续迁移，以阐明核内mRNP加工。相互作用可能是由rna结合蛋白hrp65介导的，hrp65是DHBS（果蝇行为，人类剪接）蛋白家族的成员，具有人类同源物。（iii）最后，我们打算以高空间和时间分辨率研究单个mRNPs通过核孔的易位。在这里，我们专注于mRNA输出动力学和构象动力学，并希望在单个mRNP水平上分析mRNA加工与核输出之间的联系。

The export of messenger ribonucleoprotein particles (mRNPs) can be regulated. This regulation probably has a strong impact on the gene expression outcome, but is poorly understood. Our aim is to work out the mechanisms of mRNP trafficking and export in an insect model system. We have recently shown that we can make single, specific, native mRNPs – the Balbiani ring (BR) 2 mRNP in salivary gland cells of Chironomus tentans larvae – visible and follow their movements in the nucleoplasm of living cells. We now plan to use this system for this tripartite project. (i) A special microscope shall be developed featuring color-corrected light sheet illumination for single molecule observation with unprecedented sensitivity, (ii) the detected discontinuous mobility of the BR mRNPs in nucleoplasmic regions devoid of chromatin shall be examined to elucidate intranuclear mRNP processing. Interactions are possibly mediated by the RNA-binding protein hrp65, a member of the DHBS (Drosophila behaviour, human splicing) protein family with human orthologs. (iii) Finally, we intend to study the translocation of single mRNPs through the nuclear pore with high space and time resolution. Here, we focus on mRNA export kinetics and conformational dynamics, and want to analyze links between mRNA processing and nuclear export at the single mRNP level.