1. Peter Y. Ji, Sandra C. Tseng, and Aseem Z. Ansari
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
∆Rai1 shows depletion of global mRNA in CMK treated cells.
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. A
+CMK B
+DMSO
Rai1
kin28is
∆Rai1 kin28is
∆Rai1 WT
∆Rai1 AS
∆Rai1 IS
Kin28 WT
Kin28 AS
Kin28 IS B
DMSO
1-NAPP-1
CMK
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.
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.
Graphs of optical density from 0 to 25 minutes.
A) kin28is grown in control (DMSO), reversible inhibitor 1-NAPP-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.
∆Rai1 kin28is
Kin28is
Growth curve of ∆Rai1 kin28is reveals longer growth initiation
Conclusions A B
∆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 kinase-inhibited yeast.
The 5’-end methylguanosine cap and its recruitment by kin28 relates to mRNA transcriptional success and cell viability.
DMSO
1-NAPP-1
CMK
DMSO
1-NAPP-1
CMK
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)
kin28
Pol II
m7Gppp cap
mRNA
Rai1
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. A B
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.
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