Schreck, Heidrun (2010): Translational control by the multi-KH domain protein Scp160. Dissertation, LMU München: Faculty of Chemistry and Pharmacy |
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Abstract
The control of mRNA translation mediated by RNA-binding proteins (RBPs) is a key player in modulating gene expression. In S. cerevisiae, the multi-KH domain protein Scp160 associates with a large number of mRNAs and is present on membrane-bound and, to a lesser extent, cytosolic polysomes. Its binding site on the ribosome is close to the mRNA exit tunnel and in vicinity to Asc1, which constitutes a binding platform for signaling molecules. The present study focused on the closer characterization of the Scp160-ribosome interaction and on the suggested function of Scp160 in modulating the translation of specific target mRNAs. Using affinity purifications, the partial RNA-dependence of the Scp160-ribosome association was confirmed. In contrast to published results, ribosome association was found to be only slightly reduced but not abolished in the absence of Asc1 or the last two KH domains. Furthermore, the putative elongation regulator Stm1 was identified as a co-purifier of Scp160. In subcellular fractionation experiments, RNA-binding mutants of Scp160 were present in the ribosome-free cytosolic fraction and therefore partially deficient in ribosome association and/or mRNP formation. However, no physiological conditions were found that equally induce a shift of wildtype Scp160 towards the cytosolic fraction. Within the scope of a translational profiling approach, microarray analyses of RNA isolated from sucrose density gradient fractions were performed and led to the identification of a set of mRNAs that shift their position within the gradients upon Scp160 depletion, indicating changes in their translation rates. Consistent with the membrane localization of Scp160, transcripts encoding secreted proteins were significantly enriched. Using immunoprecipitation and subsequent quantitative real-time PCR (qRT-PCR), the interaction of Scp160 with a subgroup of the identified targets was confirmed and it was shown that their binding is dependent on the conserved GXXG motifs in the two C-terminal KH domains of Scp160. Furthermore, data were obtained indicating that Scp160 can act as a translational activator on some of its target mRNAs, probably on the level of translation elongation. Finally, first evidence was provided that the translational misregulation of specific target transcripts may be involved in the polyploidization that is a hallmark of Scp160-deprived cells. In summary, these data substantiate the assumption that Scp160 is involved in translational regulation of a specific, functionally related subset of mRNAs. This finding is in good accordance with the emerging view that RBPs co-regulate multiple transcripts in order to allow faster adaptation to environmental changes.
Item Type: | Theses (Dissertation, LMU Munich) |
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Subjects: | 500 Natural sciences and mathematics 500 Natural sciences and mathematics > 540 Chemistry and allied sciences |
Faculties: | Faculty of Chemistry and Pharmacy |
Language: | English |
Date of oral examination: | 8. July 2010 |
1. Referee: | Jansen, Ralf-Peter |
MD5 Checksum of the PDF-file: | dc0132af70b0066dfe917912addf2eaf |
Signature of the printed copy: | 0001/UMC 18723 |
ID Code: | 11788 |
Deposited On: | 04. Aug 2010 07:47 |
Last Modified: | 24. Oct 2020 04:50 |