New Insights Into Intranuclear mRNP Dynamics

Abstract

Messenger ribonucleoprotein particles (mRNPs) move randomly within the nucleoplasm on their way to the nuclear pore [1]. In interphase the decondensed chromatin of mammalian cells largely governs the structure of the intranuclear environment and therefore has a strong impact on mRNP mobility. The salivary gland cell nuclei of Chironomus tentans harbor giant polytene chromosomes, where chromatin is compact and surrounded by extended regions of nucleoplasm void of chromatin. This allows studying mRNP mobility under conditions, which presumably correspond to that of interchromatin channels in mammalian cell nuclei. In this domain we examined the intranuclear movement of a specific endogenous mRNP, the BR2 mRNP, by high-speed single molecule fluorescence microscopy [2]. mRNP dynamics could be characterized by four diffusion coefficients, 0.015μm2/s, 0.23μm2/s, 0.7μm2/s and 3.7μm2/s, respectively.We now used 2’-O-Me-RNA oligonucleotides and molecular beacons to label the mRNPs in vivo, and studied the mobility of the BR2 mRNPs by complementary state-of-the-art fluorescence microscopy techniques: single molecule tracking, FCS, line scanning FCS and raster image correlation spectroscopy. These quantitative methods revealed that mobility components >3μm2/s were likely due to unbound oligonucleotides. The remaining components were shown to be typical for BR2 mRNP movement with all techniques and labelling approaches used.The BR2 mRNPs moved in a discontinuous manner in the chromatin-free nucleoplasm. That reflects transient interactions between diffusing BR2 mRNPs and submicroscopic intranuclear structures not containing chromatin. To reveal the nature of the transient mRNP immobilization we examined the impact of the nuclear actin-binding protein hrp65-2, as well as hrp65-1 on mRNP mobility. Altogether, our experiments provided a comprehensive view on the intranuclear trafficking of native mRNPs.[1] Gorski SA, Dundr M, T. Misteli. CurrOpinCellBiol. 2006 Jun; 18(3):284-90.[2] Siebrasse JP, Veith R, Dobay A, Leonhardt H, Daneholt B, U. Kubitscheck. PNAS. 2008 Dec 23; 105(51):20291-6.