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

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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.