The transcriptional coactivator PC4/Sub1 has multiple functions in RNA polymerase II transcription

Abstract

Transcription and processing of mRNA precursors are coordinated events that require numerous complex interactions to ensure that they are successfully executed. We described previously an unexpected association between a transcription factor, PC4 (or Sub1 in yeast), and an mRNA polyadenylation factor, CstF-64 (Rna15 in yeast), and provided evidence that this was important for efficient transcription elongation. Here we provide insight into the mechanism by which this occurs. We show that Sub1 and Rna15 are recruited to promoters and present along the length of several yeast genes. Allele-specific genetic interactions between SUB1 and genes encoding an RNA polymerase II (RNAP II)-specific kinase (KIN28) and phosphatase (FCP1) suggest that Sub1 influences and/or is sensitive to the phosphorylation status of elongating RNAP II. Remarkably, we find that cells lacking Sub1 display decreased accumulation of Fcp1, altered RNAP II phosphorylation and decreased crosslinking of RNAP II to transcribed genes. Our data provide evidence that Rna15 and Sub1 are present along the length of several genes and that Sub1 facilitates elongation by influencing enzymes that modify RNAP II.

Figure 1

Sub1 and Rna15 are recruited to promoters and remain chromatin associated during transcription elongation. ChIP analysis of HA-tagged Rpb3, Kin28, Rna15 and Sub1 strains to test the presence of these proteins in promoter (5′), coding (CD) and 3′ regions of the ACT1 and ADH1 genes. (A) Schematic diagram of the ACT1 and ADH1 genes: open boxes represent open reading frames and black bars PCR products. (B) ChIP analyses of HA-Rpb3 and HA-Kin28, which serve as positive and negative controls for proteins associated with both 5′ and 3′ or only with 5′ regions, respectively. Primers that amplified nontranscribed regions (HMR and V) were used as background controls. (C) ChIP analysis of HA-Rna15 and HA-Sub1: the left panel shows Rna15 and Sub1 at 5′ and 3′ ends of ACT1, the middle panel at 5′ and coding region of ADH1 and the right panel 5′ regions of ACT1, ADH1, PYK1 and RPS5, all four in RNA15 background. Numbers below gels represent adjusted fold over background (see Materials and methods). (D) ChIP analysis of HA-Rna15 from wild-type (SUB1) and mutant (Δsub1) cells. Numbers on the y-axis of the graph represent the percentage of Rna15 localized to 5′ and 3′ regions of ACT1 in the Δsub1 mutant relative to wild-type cells, where Rna15 localization in wild type is considered as 100%.

Figure 2

Sub1 is associated with the transcription complex throughout elongation. ChIP analysis of 5′, coding and 3′ sequences of the PMA1 gene using an HA-Sub1 expressing strain is shown. Primers that amplified HMR were used as background control. The bottom panel shows a schematic diagram of PCR primers used, and the black bars represent PCR products. Numbers below the gels were calculated as described in Figure 1 and Materials and methods.

Figure 3

Sub1 function is connected with CTD phosphorylation. We generated kin28 Δsub1 double mutant strains by crossing a Δsub1 strain with different kin28 alleles: (A) Δsub1 × kin28-T17D (YSB592) cross; (B) Δsub1 × kin28-K36A (YSB609) cross; (C) Δsub1 × kin28-T162A (YSB595) cross. Parental strains (Δsub1, kin28-T17D and kin28-T162) and double mutants were streaked on rich medium plates at 25°C (A) or at 25°C (B) and 37°C (C).

Figure 4

Rna15 and Ceg1 interact genetically. We crossed ceg1-250 strains (YSB625 or YSB491) with Δsub1 and rna15-1 strains. Single and double mutants from both crosses were streaked on rich medium plates at 25°C (left panel) and grown in liquid medium to perform the growth curve (right panel).

Figure 5

SUB1 deletion increases the slow growth defect of fcp1-1 mutant cells. (A) The upper panel is a schematic representation of Fcp1, showing the phosphatase motif (177 LVVDLDQTII 186) inside the FCP homology region (FCP Hom), the carboxy-terminal domains involved in the interaction with other components of the transcriptional machinery (TFIIB, TFIIF) (represented as R74) and the BRCT domain (Kobor et al, 2000). The lower panel shows the growth defects on rich medium for the fcp1 mutants (fcp1-1 and fcp1-3) at 25 and 37°C; Fcp1 mutated residues are indicated. (B, C) Two fcp1Δsub1 double mutant strains were generated by crossing a Δsub1 strain with either fcp1-1 strain (YMK20) or fcp1-3 (YMK28). Growth of double mutants and the parental strains (fcp1-1 or fcp1-3 and Δsub1) was tested by streaking them on rich medium at 25°C (B) or at 25 and 37°C (C).

Figure 6

Sub1 is a regulator of Fcp1 and influences RNAP II accumulation. (A) Extracts prepared from isogenic Δsub1 and wild-type (SUB1) cells, YOC07 and YOC06 respectively, were subjected to Western blot and probed with anti-RNAP II, anti-Fcp1 and anti-HA (to detect Kin28p) antibodies. PGK and Cox3 proteins were used as loading controls. (B) Northern blot analysis of total RNAs prepared from Δsub1 and SUB1 cells (left panel), and the corresponding agarose gel showing equal amounts of RNA loaded (right panel). (C, D) Western blots of samples from four spores (same as those used in Figure 5B), grown at 30°C and then for 1 h at 37°C, were probed with three different anti-RNAPII CTD antibodies, 8WG16, H14 and H5 (as indicated). (E) Same samples as in panels C and D, but probed with anti-Fcp1 and anti-PGK (control) antibodies.

Figure 7

SUB1 deletion affects RNAP II and Fcp1 crosslinking to promoter and coding regions. ChIP analysis of Fcp1 (A) and RNAP II (B, C) in SUB1 and Δsub1 isogenic cells, using anti-Fcp1 (A), 8WG16 (B) or H5 (C) antibodies. PCR primers for ACT1 and ADH1 were the same as those used in Figure 1. Numbers under gels and/or graphs compare relative crosslinking of RNAP II or Fcp1 to the 5′ and 3′ of the ACT1 gene and 5′ and coding region (CD) of the ADH1 gene in Δsub1 cells relative to wild-type (SUB1) cells, taken as 100.