Rai1 depletion does not restore viability in yeast in kin28 defective strains
Peter Y. Ji, Sandra C. Tseng, and Aseem Z. Ansari
UW Department of Biochemistry, University of Wisconsin-Madison
Background
Kin28is kinase inhibition retards cell growth
Kin28, an enzyme that possesses many roles in DNA transcription,
initiates the elongation phase of polymerase II and recruits capping
enzymes to process nascent RNA shortly after RNA transcripts are
produced (1). These activities are achieved through the
phosphorylation of residues on the carboxyl-terminal domain
(CTD) of polymerase, a large subunit that undergoes many
modifications throughout transcription (2). Although the
requirement of kin28 in efficient transcription is not proven,
chemical inhibition of kin28 decreases the growth rate of yeast in
kin28-analog sensitive strains (Tseng et. al, 2012). Since the loss of
kin28 function may cause a decrease in overall transcriptional
activity, we wished to explore an approach to synthetic rescue
through restoring transcriptional efficiency. Synthetic rescue was
attempted by testing genetically modified strains that lacked an
exonuclease, Rai1, when kin28-inhibited (3).
These results demonstrate that kin28 function cannot be
dispensed by the removal of exonuclease Rai1.
+CMK
A
∆Rai1 shows depletion of global
mRNA in CMK treated cells.
+DMSO
B
∆Rai1 WT
A
1
1
Kin28 WT
0.8
0.8
Kin28 AS
0.6
0.6
0.4
0.4
0.2
0.2
1-NAPP-1
∆Rai1 IS
Kin28 IS
A)
Drop test of kin28 WT, as, is strains showed less growth
following CMK and 1-NAPP-1 treatment to DMSO control.
Exonuclease dropout did not result in significant cell rescue in
CMK treated cells.
∆Rai1 kin28 WT, as, is strains are less healthy than w/ Rai1
strains, regardless of kinase inhibition.
0
Rps13
1.2
0.6
0.8
CMK
0.6
0.4
0.4
0.2
0.2
0
Pol II
mRNA
Pol II
mRNA
Rai1
5
10
15
Time (hr)
kin28
m7Gpp
p cap
A) Kin28 recruits a capping complex onto nascent mRNA via Ser5-P
marks on the CTD. The 5’end cap protects mRNA from exonuclease
degradation. B) kin28 inhibition results in the action of Rai1, an
exonuclease that acts quickly after RNA synthesis to degrade uncapped
mRNA transcripts.
•
CMK
•
•
0
0
B
1-NAPP-1
OD600
OD600
0.8
•
DMSO
1
1-NAPP-1
20
0
5
10
15
20
Time (hr)
Graphs of optical density from 0 to 25 minutes.
A) kin28is grown in control (DMSO), reversible inhibitor 1NAPP-1 and irreversible inhibitor CMK. Addition of CMK
potently sequesters cell growth. Doubling times of kin28is of
OD600 from initial OD of 0.2 were 3.9 minutes and 5.0 min for
DMSO and 1-NAPP-1 respectively. B) The same methods were
applied as kin28is with the ∆Rai1 mutation. Mutants showed
decreased cell growth and growth phase initiation. In DMSO
control, mutants had a 5.8 minute doubling time, compared
to 3.9 minutes in non-mutant kin28is. Application of
reversible 1-NAPP-1 drastically hindered cell division to a
doubling time of 17 minutes. This suggests that ∆Rai1
knockouts contribute to a low-growth phenotype especially
under conditions of kin28 deprivation through the
accumulation of uncapped RNA transcripts.
Hsp12
0
Rps13
Adh1
Hsp12
Conclusions
1.2
DMSO
1
Adh1
CMK
mRNA expression levels were normalized against DMSO levels in ∆Rai1 and
w/ Rai1 strains. B) Relative levels of mRNA were unaffected/not raised by
the lack of Rai1 upon inhibition in kin28is strains. mRNA was substantially
depleted by kin28 inhibition in both ∆Rai1 and kin28is strains. Recovery of
mRNA in Hsp12, significantly in 1-NAPP-1, may be an experimental error.
Growth curve of ∆Rai1 kin28is reveals longer growth
initiation
∆Rai1 kin28is
Kin28is
A
B
kin28
DMSO
1.2
Kin28 phosphorylates Ser5 and Ser7 on the CTD at the
transcription start site on hypophosphorylated RNA Polymerase II,
stimulating transcription and the recruitment of other CTD
kinases. (Adapted from Hinnebusch et al., 2012)
∆Rai1
kin28is
B
1.2
∆Rai1 AS
B)
A
Rai1
kin28is
•
∆Rai1 neither restores amounts of stable
transcripts nor results in synthetic rescue
Global mRNA levels in ∆Rai1 kin28is over
kin28is do not increase in +CMK cells.
Kin28 function is important in
transcriptional and translational efficiency.
Decrease in cell viability couples with low
transcriptional efficiency in kinaseinhibited yeast.
The 5’-end methylguanosine cap and its
recruitment by kin28 relates to mRNA
transcriptional success and cell viability.
Further Directions
•
•
•
Analyze nascent RNA for levels of m7G capped
transcripts in relation to degree of kin28is inhibition
using western blotting.
Analyze the efficiency of Rai1 mRNA degradation in
kin28-inhibited strains by residual levels of un-capped
transcripts.
Measure the accumulation rate of incompletely
capped mRNA in ∆Rai1, +CMK kin28is strains by
comparison of nascent RNA levels against global RNA.
References:
1)
2)
3)
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