Nuclear export of single native mRNA molecules observed via light sheet fluorescence microscopy and transcriptional regulation of BR2.1 during heat-shock

Abstract

Eucaryotes store most of their genetic information in the nucleus. Parts of this information encode the amino acid sequence of proteins. To synthesize a protein according to the nucleotide sequence, first the corresponding DNA-sequence is transcribed by RNA-Polymerase II to mRNA. Subsequently ribosomes translate the mRNA into the correct amino acid sequence. In eucaryotes the ribosomes are localized in the cytoplasm and are separated from the nucleus by the nuclear envelope. On the one hand separation of transcription and translation enables eucaryotes to process the transcript post-transcriptionally, on the other it requires a transport of the mRNA from the nucleoplasm into the cytoplasm. The nucleoplasm is interconnected with the cytoplasm by nuclear pore complexes. Most of the nucleo-cytoplasmic trafficking is facilitated through the nuclear pore complexes. Messenger RNA is exported into the cytoplasm through the nuclear pore complexes, too. During transcription the nascent mRNA is bound by several proteins which are essential e.g. for mRNA processing and export. The complex of the mRNA and its associated proteins is called an mRNP-particle. Fully processed mRNP-particles are able to cross the permeability barrier of nuclear pore complexes. In this thesis the kinetics of the mRNA-export were measured in salivary gland cells of <em>C. tentans</em> at the single molecule level.<br /> Therefore, mRNA was labeled by Hrp36, which was bacterially expressed and subsequently covalently linked to a fluorescent dye. Hrp36 associates cotranscriptionally with the nascent mRNA and is part of the mRNP-particle. After microinjection, labeled Hrp36 is transported into the nucleus, via its endogenous M9-shuttle domain. As all mRNP-particles, also the labeled ones, diffuse through the nucleus after transcription is finished and can be imaged by advanced fluorescence microscopy.<br /> In this thesis it is shown that the kinetics of the mRNA-export across the nuclear prore complexes follow a broad distribution in the range of 20ms to seconds. Furthermore, only 30% of all mRNP-particles are exported after they engaged an NPC. Fitting the mRNA-export kinetics with a bimodal gamma distribution revealed average export times of t_1exp = 76ms, which is governed by multiple rate limiting steps and t_2exp = 158ms, which is governed by just a single rate limiting step. Therefore, the translocation of the mRNA across the nuclear pore complex is not rate limiting for protein-biosynthesis which takes on average several minutes. Trajectory analysis of export events =300ms, showed that the mRNA were localized mainly in the nuclear basket during the export process. Here proteins are localized which are crucial for the mRNP-particle quality control. These proteins bind mRNP-particles, which are only partially processed, and thereby inhibit their translocation through the nuclear pore complex until their processing is completed. Assuming that the general reaction scheme is the same for all mRNP-particles and considering the fact that these slow export events show only a single rate limiting reactions step, this export events presumably correspond to mRNP-particles, whose processing were not finished. <br /> In addition to the mRNP-particle export kinetics, the Dbp5 interaction kinetics with the nuclear pore complexes were measured. Dbp5isaRNA-helicase, which is essential form RNP- particle export. It is assumed that Dbp5 removes the transport receptors from the mRNA via its helicase activity and thereby inhibit the translocation of mRNA back into the nucleus. The interaction kinetics of Dbp5 showed two interaction times (t_{1Dbp5 200Hz} = 26ms & t_{2Dbp5 200Hz} = 240ms). Due to the low number of observations, the interaction times gained by fitting the data with a bimodal gamma distribution showed a high uncertainty This makes a comparison of this results with the observed mRNA-export kinetics not advisable. <br /> In the second part of the thesis a so far unknown regulation mechanism of transcription was studied. First hints to this mechanism were observed by a control experiment during the examination of the mRNA-export kinetics. Transcription can be subdivided into the four stages of initiation, early elongation, stable elongation and termination. It was previously believed that after transition into stable elongation the transcription process is either completed or terminated prematurely. The results of this thesis give evidence that the transcription process in salivary gland cells of <em>C. tentans</em> can be halted temporally at the stage of stable elongation by applying a heat-shock to the larvae. The halted transcription processes can be resumed after heat-shock is released. <br /> Since RNA-polymerase II is highly conserved throughout eucaryotes, it seems very likely that this regulatory mechanism is not limited to <em>C. tentans</em> . The transcription halt during stable elongation described here, shows that eucaryotes have a more direct and far-ranging access to transcription as believed. This direct control of transcription significantly increases the temporal dynamic of transcriptional regulation.