Functional Analysis of the RNA Polymerase II C-terminal Domain Kinase Ctk1 in the Yeast Saccharomyces cerevisiae

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.