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. 2025 Oct 8;15(1):35162.
doi: 10.1038/s41598-025-19021-7.

The yeast checkpoint kinase Mec1p functions in transcription termination by facilitating recruitment of Pcf11p and regulating the torpedo exonuclease Rat1p

Riddhi Mehta  1 , Fatema Zohra Sadia  1 , Suprataptha U Reddy  1 , Ivana Vancurova  1 , Ales Vancura  2
Affiliations

The yeast checkpoint kinase Mec1p functions in transcription termination by facilitating recruitment of Pcf11p and regulating the torpedo exonuclease Rat1p

Riddhi Mehta et al. Sci Rep. .

Abstract

Transcription cycle of RNA polymerase II (RNAPII) features three stages: initiation, elongation, and termination. Termination, closely linked with pre-mRNA 3' processing, dissociates RNAPII from DNA and releases the nascent RNA transcript. Efficient termination is required for maintaining a pool of RNAPII that is available for re-entry into new transcription cycle. Previous results showed that inactivation of checkpoint kinase Mec1p in the absence of exogenous genotoxic stress downregulates the efficiency of transcription termination and reduces the efficiency of pre-mRNA cleavage at the polyadenylation (pA) sites. We report here that Mec1p impacts transcription termination at two distinct steps. Mec1p promotes recruitment of Pcf11p, a subunit of the cleavage factor IA (CF IA), to 3' ends of genes, and regulates the activity of torpedo exonuclease Rat1p. Deletion of Mec1p or mutations that prevent activation of Mec1p partially suppress both transcription termination defects as well as rRNA and snoRNA processing defects in rat1-1 cells. These results suggest that Mec1p regulates features of Rat1p function that are shared by termination of RNAPII transcription and rRNA and snoRNA processing and imply that the kinase activity of Mec1p downregulates Rat1p function. Cumulatively, our results reveal a new role for checkpoint kinase Mec1p in transcription termination and regulation of the torpedo exonuclease Rat1p.

Keywords: Saccharomyces cerevisiae; Checkpoint control; Checkpoint kinase Mec1p; Pcf11p; Torpedo exonuclease Rat1p; Transcription termination; pre-mRNA cleavage.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Mec1p is required for recruitment of Pcf11p to 3’ ends of PMA1 and PYK1. (A) Occupancy of Pcf11p at the indicated positions of PMA1 and PYK1 in PCF11-myc (RM124) and mec1∆sml1∆ PCF11-myc (RM126) cells. The top diagram of each gene shows schematic representation of the primers used in ChIP analysis. The same numbers are used in later ChIP figures. The results were calculated as fold increase in Pcf11p-myc or RNAPII occupancy at the particular position in comparison with the negative control. (B, C) Occupancies of Ser2P RNAPII and RNAPII, and ratio of Ser2P RNAPII/RNAPII occupancies across PMA1 (B) and PYK1 (C) genes in (WT) (W303-1a) and mec1∆sml1∆ (SN755) cells. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA and Tukey’s test. (D) Pcf11p-myc levels in wild-type (WT) (W303-1a; negative control), PCF11-myc and mec1∆sml1∆ PCF11-myc cells. Western blot was performed three times, and representative results are shown.
Fig. 2
Fig. 2
Inactivation of MEC1 suppresses growth defect and pre-mRNA processing defects in cells with reduced expression of PCF11 and RNA14, respectively. (A) Inactivation of MEC1 suppresses growth defect of tetO7-PCF11 cells. Tenfold serial dilutions of wild-type (WT) (W303-1a), mec1∆sml1∆ (SN755), tetO7-PCF11 (SN860), tetO7-PCF11mec1∆sml1∆ (SN865), tetO7-RNA14 (SN890), tetO7-RNA14mec1∆sml1∆ (SN892), tetO7-RNA15 (SN889), tetO7-RNA15mec1∆sml1∆ (SN877), tetO7-YSH1 (SN899), and tetO7-YSH1mec1∆sml1∆ (SN898) cells were spotted onto YPD plates and YPD plates containing doxycycline at 30–100 µg/ml and grown for 48 h (YPD, YPD + 30 µg/ml) or 72 h (YPD + 100 µg/ml). (B–E) Inactivation of MEC1 suppresses pre-mRNA processing defects in cells with reduced expression of RNA14 and RNA15. All strains were grown in YPD medium containing Doxycycline at 30 µg/ml. Ratios of mRNA levels at positions B and A (B, D) and C and A (C, E) of PMA1 (B, C) and PYK1 (D, E). Positions A, B, and C in PMA1 and PYK1 are also used in later figures. The results for each primer are normalized to RDN25 RNA and are shown as arbitrary units (A.U.). The mean for WT cells was set as 100 A.U. The data are means ± SD from four biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparisons test.
Fig. 3
Fig. 3
Inactivation of MEC1 suppresses transcription termination defects in cells with reduced expression of PCF11 or RNA14. (A, C) RNAPII occupancies across (A) PMA1 and (C) PYK1 genes in wild type (WT) (W303-1a), mec1∆sml1∆ (SN755), tetO7-PCF11 (SN860), tetO7-PCF11mec1∆sml1∆ (SN865), tetO7-RNA14 (SN890), and tetO7-RNA14mec1∆sml1∆ (SN892). All strains were grown in YPD medium containing Doxycycline at 30 µg/ml. The results were calculated as fold increase in RNAPII occupancy at the particular position in comparison with the negative control. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA and Tukey’s test. (B) Ratios of RNAPII occupancies at positions 6 and 3 of PMA1 and (D) positions 4 and 3 of PYK1. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparison test.
Fig. 4
Fig. 4
Inactivation of MEC1 does not suppress transcription termination defects of pcf11-2 and pcf11-9 cells. (A–D) Ratios of mRNA levels at positions B and A (A, C) and C and A (B, D) of PMA1 (A, B) and PYK1 (C, D) in wild type (WT) (W303-1a), mec1∆sml1∆ (SN755), pcf11-2 (DBY593), mec1∆sml1∆ pcf11-2 (RM289), pcf11-9 (DBY585), and mec1∆sml1∆ pcf11-9 (RM274) strains. The positions of primers are the same as in Fig. 2. The results for each primer are normalized to RDN25 RNA and are shown as arbitrary units (A.U.). The mean for WT cells was set as 100 A.U. The data represent means ± SD from four biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparisons test. (E, G) RNAPII occupancies across PMA1 and PYK1 genes in the same strains. The positions of primers are the same as in Fig. 1. The results were calculated as fold increase in RNAPII occupancy at the particular position in comparison with the negative control. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA and Tukey’s test. (F) Ratios of RNAPII occupancies at positions 6 and 3 of PMA1 and (H) positions 4 and 3 of PYK1. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparison test.
Fig. 5
Fig. 5
Inactivation of MEC1 suppresses pre-mRNA processing and transcription termination defects of rat1-1 cells. (A–D) Ratios of mRNA levels at positions B and A (A, C) and C and A (B, D) of PMA1 (A, B) and PYK1 (C, D) in wild type (WT) (W303-1a), mec1∆sml1∆ (SN755), rat1-1 (SN802), rat1-1 rat1-1mec1∆sml1∆ (RM121), xrn1∆ (MB114), xrn1∆mec1∆sml1∆ (RM120), rat1-1xrn1∆ (SN800), and rat1-1xrn1∆mec1∆sml1∆ (RM123) strains. The positions of primers are the same as in Fig. 2. The results for each primer are normalized to RDN25 RNA and are shown as arbitrary units (A.U.). The mean for WT cells was set as 100 A.U. The data are means ± SD from four biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparisons test. (E, G) RNAPII occupancies across (E) PMA1 and (G) PYK1 genes in wild type (WT), mec1∆sml1∆, rat1-1, and mec1∆sml1∆rat1-1 strains. The positions of primers are the same as in Fig. 1. The results were calculated as fold increase in RNAPII occupancy at the particular position in comparison with the negative control. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA and Tukey’s test. (F) Ratios of RNAPII occupancies at positions 6 and 3 of PMA1 and (H) positions 4 and 3 of PYK1. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparison test.
Fig. 6
Fig. 6
Inactivation of MEC1 suppresses ribosomal RNA and snoRNA processing defects of rat1-1 cells. (A, D) Simplified diagram of 5.8 S, 25 S rRNA, and snR190 processing. The positions of primers used for RT-qPCR analysis is indicated by the solid bar above 5.8 S, 25 S rRNA, and snR190 sequences. Levels of unprocessed (B) 5.8 S rRNA, (C) 25 S rRNA, and (E) snR190 RNA in wild type (WT) (W303-1a), mec1∆sml1∆ (SN755), rat1-1 (SN802), rat1-1mec1∆sml1∆ (RM121), xrn1∆ (MB114), xrn1∆mec1∆sml1∆ (RM120), rat1-1xrn1∆ (SN800), and rat1-1xrn1∆mec1∆sml1∆ (RM123) strains. The results for each primer are normalized to RDN25 RNA and are shown as arbitrary units (A.U.). The mean for WT cells was set as 100 A.U. The data are means ± SD from four biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparisons test.
Fig. 7
Fig. 7
Inactivation of MEC1 does not promote recruitment of Rat1p and Rat1-1p to 3’ ends of PMA1 and PYK1. (A) Rat1p-TAP and Rat1-1p-TAP occupancies across PMA1 and PYK1 genes in RAT1-TAP (RM345), mec1∆sml1∆RAT1-TAP (RM347), rat1-1-TAP (SN802), and mec1∆sml1∆rat1-1-TAP (RM121) cells. The results were calculated as fold increase in Rat1p-TAP or Rat1-1-TAP occupancy at the particular position in comparison with the negative control. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA and Tukey’s test. (B) Rat1p-TAP and Rat1-1p-TAP levels in wild-type (WT) (W303-1a; negative control), RAT1-TAP, mec1∆sml1∆RAT1-TAP, rat1-1-TAP, and mec1∆sml1∆rat1-1-TAP cells. Western blot was performed three times, and representative results are shown.
Fig. 8
Fig. 8
Suppression of transcription termination and ribosomal RNA and snoRNA processing defects in rat1-1 cells requires activation of Mec1p. (A) Levels of unprocessed 5.8 S rRNA, 25 S rRNA, and snR190 RNA in wild type (WT) (W303-1a), rat1-1xrn1∆ (SN800), rad17∆ (W152211B), rat1-1xrn1∆rad17∆ (RM317), rad24∆ (W151917B), rat1-1xrn1∆rad24∆ (RM324), rad9∆ (SJ027), and rat1-1xrn1∆rad9∆ (RM321). (B, C) Inactivation of RAD17 suppresses pre-mRNA processing defects of rat1-1 and rat1-1 xrn1∆ cells. Ratios of mRNA levels at positions B and A (B) and C and A (C) of PMA1. The positions of primers are the same as in Fig. 2. (D) RNAPII occupancies across PMA1 in wild type (WT), rad17∆, rat1-1, and rat1-1rad17∆ strains. The positions of primers are the same as in Fig. 1. The results were calculated as fold increase in RNAPII occupancy at the particular position in comparison with the negative control. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA and Tukey’s test. (E) Ratios of RNAPII occupancies at positions 6 and 3 of PMA1. The data are means ± SD from four biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Bonferroni’s multiple comparison test.
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