Affinity purification of mRNA-protein complexes from Saccharomyces cerevisiae

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Dokumentart: PhDThesis
Date: 2016-05
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biochemie
Advisor: Jansen, Ralf-Peter (Prof. Dr.)
Day of Oral Examination: 2014-07-17
DDC Classifikation: 500 - Natural sciences and mathematics
Keywords: RNS
Other Keywords: Affinitätsaufreinigung
RNA-binding protein
affinity purification
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A functional mRNA never exists inside a cell in a naked form but it is always bound by proteins. RNA-binding proteins (RBPs) regulate every aspect of mRNA function, including splicing, export, translation and stability. However, the composition and function of many mRNA-protein complexes (mRNPs) is poorly understood. This is due to the complexity of mRNP biogenesis as well as to technical limitations. I have established a method of mRNP affinity purification from Saccharomyces cerevisiae. In order to isolate the mRNA of interest the mRNA is tagged with MS2 stem-loops (MS2L) before the 3′ UTR. The tagged mRNA is co-expressed with MS2 coat protein (MS2CP) fused to protein A, thus allowing in vivo binding of the MS2L tag to MS2CP. mRNPs are affinity-purified using IgG coated magnetic beads via Protein A-IgG interaction. Quantitative analysis of MS2L-tagged RNA co-purifying proteins is achieved using SILAC metabolic labelling technique in combination with sample analysis by liquid chromatography-tandem mass spectrometry. About 60-70% of proteins co-purifying with two mRNAs encoding for glycolytic enzymes, PGK1 and ENO2, have a well established role in mRNA biology. The repertoire of the proteins co-purifying with the analysed MS2L-tagged mRNAs reflects both nuclear and cytoplasmic steps of mRNA life cycle. Many of the enriched proteins have a role in mRNA translation or decay, suggesting that a large subpopulation of MS2L-tagged PGK-1 or ENO2-containing mRNPs is engaged in these two processes. Besides proteins with a well established role in mRNA biology multiple unexpected proteins co-purified with MS2L-tagge PGK1 and ENO2. Our results suggest that ribosome biogenesis factors, tRNA-modifying enzymes and some metabolic enzymes may play a role in mRNA biology as components of mRNPs. In addition, our results hint at the possibility that gycolytic enzymes may co-translationally associate into multi-enzyme complexes.

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