In the eukaryotic cell, the nuclear envelope separates the nucleoplasm from the cytoplasm. The nuclear pore complex (NPC) forms the conduit that regulates the exchange of macromolecules between these compartments. Import and export of protein and RNA through the NPCs are highly regulated and follow several different pathways. Messenger RNAs (mRNAs) are exported from the nucleus only after extensive processing and assembly into ribonucleoprotein particles (RNPs). Studies in yeast have identified many components involved in mRNA export. These include nuclear pore proteins, export receptors and components in the nucleus that couple formation of mRNPs with translocation through the pores. When I started my PhD work, I performed a synthetic lethal (sl) screen to investigate the role of Gle2 in mRNA export. Gle2 was a proposed to be an mRNA export factor. Thus, the aim has been to gain an understanding of the relationship between Gle2 and the Mex67-mediated mRNA export pathway. I could show that GLE2 is synthetic lethal with the mRNA export factors Sac3 and Mex67, with importins a and b, and with several nucleoporins, which are subunits of distinct subcomplexes of the NPC. This part of my studies indicated that the function of Gle2 is not restricted to nuclear export and suggested a more general role of Gle2 in bidirectional transport through the nuclear pore complexes. To investigate the Mex67-mediated mRNA export pathway, in the second part of my studies, I performed a synthetic lethal screen with SUB2, an intranuclear factor which in our lab was found to act in mRNA export. Initial work suggested that Sub2 was a splicing factor. The sl screen I performed revealed a genetic link between SUB2 and the THO complex, which is involved in transcription elongation. These data contributed to the identification of a novel conserved complex called TREX (transcription/export), formed by the export factors Sub2 and Yra1, a previously unknown factor, Tex1, and the THO complex. Thus, the TREX complex couples transcription elongation and mRNA export. To further characterize the TREX, I analyzed the genetic interactions of two components of the THO complex, THO2 and THP2. The import receptor MTR10 was found to be synthetic lethal with SUB2 and THP2. In addition, I found that Sub2-GFP and Thp2-GFP are mislocalized in MTR10 mutants, indicating a role of Mtr10 as import factor for components of the TREX complex. Furthermore, THO2 and THP2 are synthetic lethal with RRP6, a component of the exosome complex, which retains and eliminates improperly 3�-end processed mRNPs, suggesting a link between transcription elongation, and RNP quality control. Finally, I analyzed the TREX complex at the biochemical level. The TREX complex was purified using a variety of methods, including tandem affinity purification (TAP) and by gel filtration. Under stringent conditions, I could purify a stable core of the TREX complex, in which Sub2 and Yra1 were partly dissociated. In collaboration with the Böttcher lab (EMBL), I studied the morphology of this complex by electron microscopy (EM). The core of TREX shows a butterfly-like shape, with two-fold symmetry and a cleft in between the two winged arms. Under less stringent conditions, the TREX complex contains stoichiometric amounts of Sub2 and Yra1. Nevertheless, the complex mostly retains a butterfly-like morphology at the EM level. In conclusion, my studies identified new connections between mRNA export and protein import at the nuclear pore, and revealed that transcription, maturation and export of mRNAs are genetically and physically coupled.
|Supervisor:||Hurt, Prof. Dr. Ed|
|Date of thesis defense:||21 September 2004|
|Faculties / Institutes:||Service facilities > Heidelberg University Biochemistry Center|
|Subjects:||570 Life sciences|
|Controlled Keywords:||Transkription <Genetik>, Genexpression, Mikroskopie, Nuklearfaktor, Transkriptionsfaktor, Zellkern, Kernproteine, Kernhülle, Kernpore|
|Uncontrolled Keywords:||mRNA Export , ElektronenmikroskopiemRNA Export , electronmicroscopie , nuclear cytoplasmic transport|