Identifizierung von Gbp2p und Hrb1p als am nukleären mRNA-Export beteiligte Proteine und Analyse der zytoplasmatischen Umstrukturierungen exportierter mRNA-Proteinkomplexe
Der nukleäre mRNA-Export ist ein komplexer Vorgang, an dem zahlreiche Proteinfaktoren beteiligt sind. Pendelnde mRNA-Bindeproteine, welche in Assoziation mit den mRNA-Molekülen den Zellkern verlassen und daraufhin re-importiert werden, sind hierbei von zentraler Bedeutung. Die vorliegende Arbeit lei...
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The nuclear messenger RNA export is a complex process with numerous protein factors participating. Shuttling mRNA binding proteins, which leave the nucleus in association with the mRNA molecules and become reimported thereafter, are of central importance. In this work, progress in understanding mRNA export could be achieved by identifying two proteins, Gbp2p and Hrb1p, as novel components of export competent mRNA-protein complexes in the yeast Saccharomyces cerevisiae. Gbp2p und Hrb1p are homologous and both contain three RNA-recognition motifs and one serin/arginine (SR)-rich domain. They are associated with mRNA in vivo and leave the nucleus in dependence on continuous RNA polymerase II transcription and mRNA export factors. The export requirements of both proteins are not exactly identical, which suggests the existence of several parallel mRNA export routes. The predominantly nuclear localization of Gbp2p and Hrb1p depends on the import receptor Mtr10p and on the SR-specific protein kinase Sky1p. Furthermore, mutations of two specific SR dipeptides of Gbp2p result in a cytoplasmic mislocalization of the protein. Interestingly, deletion of MTR10 only also causes a significant increase in poly(A)+ RNA binding of Gbp2p. Although GBP2 and Hrb1 are not essential for normal cell growth, overexpression of GBP2 is toxic and causes a nuclear retention of bulk poly(A)+ RNA. Together, the presented results show an involvement of Gbp2p and Hrb1p in nuclear mRNA export. Being non-essential, both proteins also have the remarkable potential to predominantly regulate the mRNA export under specific conditions. In the second part of this work, a comprehensive analysis of the cytoplasmic rearrangements of exported mRNA-protein complexes prior to and during translation was carried out. Striking differences in the dissociation behavior of the shuttling mRNA binding proteins could be observed. While only a few molecules of Hrp1p, Nab2p, and Mex67p cosediment with active translation complexes, significant amounts of the SR-rich proteins Gbp2p, Hrb1p, and Npl3p and of the RNA-helicase Dbp5p are associated with ribosome-bearing mRNA molecules. Interestingly, a prolonged association of the export factor Npl3p with polysomes results in a translational defect, indicating that Npl3p can function as a negative translational regulator. Moreover, using sucrose density gradient analyses, it was shown that the import receptor Mtr10p, but against prior assumptions not Sky1p, is involved in the cytoplasmic mRNA dissociation of Npl3p.