Deciphering the RNA polymerase II CTD code in fission yeast

Abstract

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats. Dynamic remodeling of the CTD, especially its serine phosphorylation pattern, conveys informational cues about the transcription apparatus to a large ensemble of CTD-binding proteins. Our genetic dissection of fission yeast CTD function provides insights to the "CTD code." Two concepts stand out. First, the Ser2 requirement for transcription during sexual differentiation is bypassed by subtracting Ser7, signifying that imbalance in the phosphorylation array, not absence of a phospho-CTD cue, underlies a CTD-associated pathology. Second, the essentiality of Ser5 for vegetative growth is circumvented by covalently tethering mRNA capping enzymes to the CTD, thus proving that capping enzyme recruitment is a chief function of the Ser5-PO(4) mark. This illustrates that a key "letter" in the CTD code can be neutralized by delivering its essential cognate receptor to the transcription complex via an alternative route.

Figure 1. Effects of CTD mutations on fission yeast viability, vegetative growth, and mating proficiency

(A) The amino acid sequences the CTDs encoded by rpb1 alleles “WT”, T4A and S5A are displayed at left with the heptad repeats aligned vertically. The alleles are named according to the amino acid substitutions introduced into all 14 consensus heptads appended to the “rump” that connects the CTD to the body of Pol II. A summary of the mutational effects on growth and mating is compiled in the Table at right. (B) Viable S. pombe strains with the indicated rpb1-CTD alleles were grown in liquid medium until A₆₀₀ reached 0.6 to 0.9. The cultures were adjusted to A₆₀₀ of 0.1 and aliquots (3 μl) of serial 5-fold dilutions were spotted on YES agar plates. The plates were photographed after incubation for 8 d at 18°C, 6 d at 20°C, 3 d at 25° or 2.5 d at 30°, 34° and 37°C.

Figure 2. S7A suppresses the requirement for Ser2 for fission yeast mating and ste11 induction

(A) Representative photomicrographs of homothallic WT, S2A and S2A+S7A cells after 24 h in mating/sporulation medium (malt). Cells were treated with DAPI and then visualized by differential interference contrast (left panels) and fluorescence (right panels) microscopy. The stars indicate examples of asci. (B) Northern blot analysis of ste11⁺ and mei2⁺ mRNA levels in exponentially growing homothallic WT, S2A and S2A+S7A cells before (YES), or 5 hr after transfer to mating medium (malt). As a control for equal loading of RNA in each lane, ribosomal RNAs (28S and 18S) stained with ethidium bromide (negative image) are shown in the bottom panel. (C) Western blot analysis of Rpb1 CTD phosphorylation status in exponentially growing heterothallic WT, S2A, S7A and S2A+S7A cells using antibody to bulk Rpb1 (8WG16, top panel) and phospho-specific antibodies against Ser7-P (middle panel) and Ser2-P (bottom panel). The positions and sizes (kDa) of marker proteins are indicated at left.

Figure 3. The lethality of S5A is bypassed by fusion of capping enzyme to the CTD

(A) The S5A-Mce1 fusion is depicted at right. rpb1-S5-MCE1 and rpb1-S5A-MCE1 cells were grown in liquid culture at 30°C and serial dilutions were spotted to YES agar. The plates were photographed after 4 d at 25°C, or 3 d at 30 and 34°C. (B) Pol II immunoblots of extracts of rpb1⁺, S5-MCE1, and S5A-MCE1 cells using 8WG16 (left panel) or anti-Ser5-P (right panel) antibodies. The positions and sizes (kDa) of marker proteins are indicated at left. (C) Guanylyltransferase activity was gauged by label transfer from [α³²P]GTP to the active capping enzymes in the extract to form covalent enzyme-[³²P]GMP adducts detectable by SDS-PAGE and autoradiography. The positions of marker proteins are as in panel B. (See also Fig. S1.)

Figure 4. S5/S5A CTD chimaeras

S. pombe strains with the indicated rpb1-CTD alleles were grown in liquid YES medium. The cultures were adjusted to A₆₀₀ of 0.1 and aliquots (3 μl) of serial 5-fold dilutions were spotted on YES agar plates. The plates were photographed after 7 d at 18°C, 6 d at 20°C, 3 d at 25°C, or 2.5 d at 30°C, 34°C, and 37°C.