Charakterisierung neuer Funktionen der mRNA-Exportfaktoren Npl3p und Dbp5p in der Translation

Durch die Kompartimentierung einer eukaryontischen Zelle erfolgen die Genexpression und die Proteinbiosynthese an verschiedenen Orten. Im Zellkern findet die Transkription der Gene in die komplementären prä-mRNA’s statt, die durch die nukleäre Prozessierung zu exportkompetenten mRNA’s reifen. Diese...

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Bibliographic Details
Main Author: Gross, Thomas
Contributors: Krebber, Heike (Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2009
Molekularbiologie und Tumorforschung
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Table of Contents: The compartmentalization between nucleus and cytoplasm is the principal defining feature of eukaryotic cells and leads to the spatial separation of transcription and translation. Transcription occurs in the nucleus resulting in the complementary pre-mRNA which is processed into an export competent mRNA. The assembly of export mediating proteins with the mRNA facilitates the transport of the mRNP complex through the nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, into the cytoplasm. Subsequently, protein synthesis occurs at the ribosomes in the cytoplasm by decoding the genetic code of the mRNA into the amino acid sequence of the encoded proteins. Numerous mRNA binding proteins are involved in the export of an mRNA into the cytoplasm. The majority of those proteins dissociates from the mRNA immediately upon their translocation. In contrast, some mRNA binding proteins like the DEAD box RNA helicase Dbp5p or the shuttling SR-protein Npl3p as shown in Saccharomyces cerevisiae remain bound to the mRNA during translation. This suggests possible functions of these proteins in translation, which were examined in this work. Genetical, cell biological, and biochemical data show that Npl3p and Dbp5p are involved in different phases of translation. In the first part of this thesis it is demonstrated by localization studies and analyses of physical interactions that Npl3p functions as a new export factor for the ribosomal pre-60S subunit. Additionally, genetic interactions of NPL3 with factors already known for their function in the export of the pre-60S subunit, XPO1, MTR2 and NMD3, confirm the transport function of Npl3p. Moreover, deletion of NPL3 (npl3Δ) leads to a reduced growth rate, which is caused by defects in translation but not by export defects of the pre-60S subunit. Those translational defects are caused by a reduced amount of monosomes (80S) due to a lower rate of 40S and 60S subunit association during the final step of translation initiation, the subunit joining. The novel role of Npl3p in supporting subunit joining during translation initiation is supported by both, genetical and physical interactions of NPL3 and/or Npl3p with factors involved in subunit joining. Further investigations demonstrate that Npl3p can form homodimers or homooligomers. Since Npl3p is associated with the exported mRNP and the pre-60S subunit, it might act by helping to connect both ribonucleoparticles through its self-association interaction and thereby promotes the stabilization of the 80S formation during subunit joining. In the second part of this thesis, an active function of Dbp5p during translation termination was characterized. A general role of Dbp5p during translation is supported by hypersensitivity of dbp5-mutants to translation inhibitors. Moreover, DBP5 interacts genetically not only with both translation termination factors SUP45 (eRF1) and SUP35 (eRF3) but also with the poly(A)-binding factor PAB1. Reporter assays show a requirement of the catalytic activity of Dbp5p for efficient stop codon recognition through Sup45p (eRF1). Additionally, Dbp5p interacts physically with Sup45p (eRF1), however not with Sup35p (eRF3) or Pab1p. Furthermore, mutants of DBP5 show a substantially reduced co-sedimentation of Sup35p (eRF3) with polysomes and a loss of the interaction between Sup45p (eRF1) and Sup35p (eRF3). This indicates an important role of Dbp5p for the recruitment of Sup35p (eRF3) into the termination complex. Thus, Dbp5p regulates the interaction of Sup45p (eRF1) and Sup35p (eRF3) in translation termination, however it does not seem to be involved in the termination process of premature termination codon (PTC)-containing mRNAs that triggers nonsense-mediated mRNA decay (NMD). In this thesis new functions of both mRNA export factors Npl3p and Dbp5p were identified and characterized during translation demonstrating the connection of both processes, mRNA-export and translation, through those proteins. This reveals on the one hand the multifunctionality of Npl3p and Dbp5p and on the other hand the efficiency in nature to regulate both cellular processes in eukaryotes with the same molecules.