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Roether, Susanne (2007): Functional Analysis of the RNA Polymerase II C-terminal Domain Kinase Ctk1 in the Yeast Saccharomyces cerevisiae. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Gene expression encompasses a multitude of different steps, starting with transcription in the nucleus, co-transcriptional processing and packaging of the mRNA into a mature mRNP, export of the mRNP through the nuclear pore and finally the translation of the message in the cytoplasm. The central coordinator for coupling of the nuclear events is the differentially phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAP II). The phosphorylation pattern of the CTD not only dictates the progression through the transcription cycle but also the recruitment of mRNA processing machineries. Coupling of transcription to mRNA export is achieved by the TREX complex, which consists in the yeast S. cerevisiae of the heterotetrameric THO complex important for transcription elongation, the SR-like proteins Gbp2 and Hrb1, and Tex1 and the mRNA export factors Sub2 and Yra1. By direct interaction with Yra1, the mRNA export receptor Mex67-Mtr2 is then recruited to the mRNP and transports the mRNP through the nuclear pore complex to the cytoplasm. In a genetic screen for factors that are crucial for TREX complex function in the living cell, Ctk1, a cyclin dependent kinase (CDK) that phosphorylates the C-terminal domain (CTD) of RNAP II during transcription elongation, was identified (Hurt et al. 2004). Surprisingly, besides the TREX components Gbp2 and Hrb1, Ctk1 co-purified ribosomal proteins and translation factors. Using sucrose density centrifugation, it could be shown that Ctk1 indeed associates with translating ribosomes in vivo, suggesting a novel function of this protein in translation. This assumption was confirmed by in vitro translation assays: loss of Ctk1 function leads to a reduction in translational activity. More specifically, Ctk1 is important for efficient translation elongation and contributes to the accurate decoding of the message. Cells depleted for Ctk1 are more sensitive towards drugs that impair translational accuracy and show an increase in the frequency of miscoding in vivo. The function of Ctk1 during decoding of the message is most likely direct, as in extracts of cells depleted for Ctk1 the defect in correct decoding of the message can be restored to wild type levels by addition of purified CTDK-I complex. An explanation for the molecular mechanism of Ctk1’s function is provided by the identification of Rps2 as a novel substrate of Ctk1. Rps2 is a protein of the small ribosomal subunit, located at the mRNA entry tunnel and known to be essential for translational accuracy. Importantly, Rps2 is phosphorylated on serine 238 by Ctk1, and cells containing an rps2-S238A mutation show an increased sensitivity towards drugs that affect translational accuracy and an increase in miscoding as determined by in vitro translation extracts. The role of Ctk1 in translation is probably conserved as CDK9, the mammalian homologue of Ctk1, also associates with polysomes. Since Ctk1 interacts with the TREX complex, which functions at the interface of transcription and mRNA export, Ctk1 might bind to the mRNP during transcription and accompany the mRNP to the ribosomes, where Ctk1 enhances efficient and accurate translation of the mRNA. This study could be an example of a novel layer of control in gene expression: the composition of the mRNP determines its translational fate, including efficiency and accuracy of translation.