3.2 RESULTS AND DISCUSSION
3.2.2 The analysis of the impact of promoter region in Cln2 oscillation
In order to understand the contribution of promoter region in oscillation waves of Cln2 we compared the behavior of Cln2 and shlGFP under the control of Cln2 promoter during the cell cycle. NS343-NS346 strains were created at the background of RV1110 (NS343, NS344) and RV1111 (NS345, NS346). In NS343-NS346 yeast strains Cln2 ORF was replaced with shlGFP-13Myc containing the short-half-life GFP (shlGFP) instead of Cln2 region, Sic1_WT or Sic1_9SP (all 9 pairs of TP phosphorylation sites mutated to SP), and 13Myc tag with two strains expressing either the Sic1_WT-13Myc or Sic1_9SP (yeast strains are listed in Table 1).
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To analyze the phosphorylation of the proteins of interest, SDS-page Western Blot experiments were performed with 6 different strains (The strains are listed in the Supple-mentary Table 1) containing 13Myc tags. The cells were arrested in G1 by α-factor, re-leased after 2-2.5 hours, and collected in the following time points: 0, 10, 20, 30, 40, 50, 60, 70 minutes after the release (Fig. 7).
Figure 7 Analysis of the influence of the Cln2 promoter in Cln2 protein levels. Time courses showing different protein levels of cln2::ShlGFP-13MYC; Cln2-13MYC,
Sic1_WT; and Cln2-13MYC, Sic1_9SP with 5 seconds exposure (A) and Cln2-13MYC, Sic1_WT; Cln2-13MYC, Sic1_9SP; at the 30 seconds exposure (B).
(A) Strains containing short-half-life-GFP (shlGFP) under the control of Cln2 promoter show high protein levels at 0 and 10 time points probably due to the longer sequence com-pared to the Cln2. It is clear that the protein levels of strains lacking Cln2_WT are at least 10 times higher compared to the background RV1110 and RV1111 strains at the same 5 seconds exposure.
(B) RV1110 (Cln2-13MYC, Sic1_WT ) strain shows normal Cln2 behavior during the cell cycle. RV1111 demonstrates the loss of protein levels caused by threonine-proline to ser-ine-proline substitution of the Sic1_WT phosphorylation sites.
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Figure 8. Band intensities of the Cln2 protein region in different mutants. om-parison of protein levels in cln2::ShlGFP-13MYC; 13MYC, Sic1_WT; and Cln2-13MYC, Sic1_9SP.
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The Cln2 promoter region has SBF and MBF transcription factors which activate the expression of native Cln2 and forming a positive feedback loop for Cln2 expression (Cross and Tinkelenberg, 1991). Cln2 also phosphorylates Sic1 and, therefore, targets it for degradation which leads to Clb5 activation (Schneider et al., 1996). The Cln2 is unable to phosphorylate yeast strain containing Sic1_9SP at the proper cell cycle stage which makes the Clb5 rapid activation impossible. Clb5, in turn, is responsible for Whi5 phosphoryla-tion inhibiting SBF and MBF transcripphosphoryla-tion factors. Our reference strain with Sic1_9SP shows that the promoter activation takes place at much lower rates. Nevertheless, the strains with the Cln2 region replaced by short-half-life-GFP were able to stabilize the pro-tein levels.
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SUMMARY
The current work can be divided into two parts. In the first part, we generated a set of multisite phosphorylation protein tags based on the Cln2 degron for the regulation of pro-tein expression in yeast cells. Based on the levels of fluorescence of GFP (was used as a reporter) in different strains, we can conclude that our phosphodegron tags can significantly reduce protein expression. Changing the length of the degron tags and the distance between the phosphosites it is possible to achieve the desired protein levels.
In the second part, we wanted to analyze the impact of transcriptional regulation in the oscillation waves of Cln2 protein during the cell cycle. To do that, we compared the behavior of Cln2 and short-half-life-GFP (shlGFP) proteins under the control of Cln2 pro-moter during the cell cycle. Based on the results of the Western blot, we can conclude that the reason for Cln2 cell-cycle oscillations is lying in the protein sequence itself, while pro-moter activity is likely responsible for the synthesis of a sufficient number of transcripts.
Further research of this subject will help to reveal the exact mechanism by which the inter-action between transcriptional and translational regulation of Cln2 oscillations occurs during the cell cycle.
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SUPPLEMENTARY MATERIALS
Table 1. Yeast strains
NS228 was used as a background strain (ordered from EuroScarf): CEN.PK-2-1C:
MATa ura3-52 trp1-289 leu2-3,112 his3∆ can1∆::cas9-natNT2.
№ Genotype Short description/transfor-mation
Constitutive expression of
EGFP fused with
NES/CRISPR-Cas9 mediated integration of a PCR fragment
Nastassia
35
NS292
pADH1-GFP-NES-4A-KanMX
Based on NS229, transformed using PCR product from
Based on RV1110 Nastassia Shtaida
RV1110 Cln2-13MYC, Sic1_WT Rainis
Venta
RV1111
Cln2-13MYC-Sic1_9SP
Based on RV1110
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Table 2. Plasmids used as PCR templates to amplify fragments for yeast trans-formation
№ Description: Strain:
Pri-mers (F/R):
Refer-ence:
pNS89 Cln2_5WT-15aa NS274, NS275 4731/4826
This study
pNS100 Cln2_5WT-12aa NS295 4731/4826
pNS101 Cln2_5WT-15aa NS267 4731/4826
pNS102 Cln2_5WT-18aa NS296 4731/4826
pNS103 Cln2_4WT-12aa NS348 4732/4826
pNS105 Cln2_4WT-18aa NS349 4732/4826
pNS108 Cln2_5WT-KKKK NS284, NS285 4724/4729 pNS109 Cln2_4WT-KKKK NS286, NS287 4719/4729
Table 3. Primers, used in this work.
Primer Sequence Tm (°C)
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The impact of Cln2 degron and Cln2 promoter on protein expression levels (title of thesis)
supervised by Nastassia Shtaida, PhD
(supervisor’s name)
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