Heptad-Specific Phosphorylation of RNA Polymerase II CTD

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olecular Cell Supplemental Information Heptad-Specific Phosphorylation of RNA Polymerase II CTD Roland Schüller, Ignasi Forné, Tobias Straub, Amelie Schreieck, Yves Texier, Nilay Shah, Tim-ichael Decker, Patrick Cramer, Axel Imhof, and Dirk Eick

A B ammalian -CTD: Predicted peptides containing heptads 3-: YSPTSPA YEPRSPGG 3 YTPQSPS 6 YSPTSPN 7 8 9 3 6 7 8 9 YSPTSPN 3 YSPTSPN YTPTSPS 6 YSPTSPN C D kd x 6 7 YTPTSPN 8 9 3 3 YSPSSPR 3 YTPQSPT 33 YTPSSPS 3 YSPSSPS 3 36 YTPTSPS 37 YSPSSPE 38 YTPTSPK 39 Trypsin YSPTSPT YSPTTPK 3 YSPTSPT YSPTSPV YTPTSPK 6 YSPTSPT 7 8 YSPTSPT 9 GST YSPTSPG YSPTSPT YSLTSPAISPDDSDEEN 3-3 39-3- 8-9 - 3 Western blot Proliferation YTPQSPTYTPSSPSYSPSSPS YTPTSPSYSPSSPEYTPTSPK YSPTSPTYSPTSPVYTPTSPK GSTYSPTSPGYSPTSPTYSLTSPAISPDDSDEEN Proliferation and cell viability of and E - - -7 8- -3-6 7-9 - 3-6-8 9-3 3-33 3-3 39- - 3-8-9 - - - -7 8- - 3-6-7 8- -3-6 7-9 3-3 3-33 3-3 39- - 3-8-9 - YSPTSPAYEPR SPGGYTPQSPS YSPTSPN YSPASPK YSPSSPK YSPLSPK AYSPLSPR YSPTSPNYTPTSPS YSPTSPNYTPTSPN YAPTSPSYSPASPSYAPSSPR YTPQSPTYTPSSPK YSPSSPS YTPTSPSYSPSSPEYTPTSPK YSPTSPTYSPTSPVYTPTSPR YSPTSPT GSTYSPTSPG YSLTSPAISPDDSDEEN 3 YSPTSPAYEPR SPGGYTPQSPS YSPTSPN A A AA AYSPTSPN YTPTSPS AYTPTSPN YSPASPK YTPQSPTYTPSSPK YSPSSPS YTPTSPSYSPSSPEYTPASPR AA YSPTSPTYSPTSPVYTPTSPK A GSTYSPTSPG YSLTSPAISPDDSDEEN rec 3 - - -7 8-9 - 3-6-8 9- - -8 9-3 3-3 39-3- 8-9 - - - -8 9- - -8 9-3- 6-8 9-3 3-33 3-3 39-3- 8-9 - YSPTSPAYEPR SPGGYTPQSPS YSPTSPN YSPASPK YSPSSPK AYSPLSPR YSPTSPNYSPTSPNYTPTSPK YSPTSPNYTPTSPN YSPSSPR YTPQSPTYTPSSPSYSPSSPS YTPTSPSYSPSSPEYTPASPR YSPTSPTYSPTSPVYTPTSPK YSPTSPT GSTYSPTSPGYSPTSPTYSLTSPAISPDDSDEEN (3E) YSPTSPAYEPR SPGGYTPQSPS YSPTSPN YSPASPK YSPASPS AYSPTSPNYTPTSPS YSPTSPNYTPTSPN YSPSSPR YTPQSPTYTPSSPK YSPSSPS YTPTSPSYSPSSPEYTPASPK YSPTSPTYSPTSPVYTPTSPK YSPTSPT GSTYSPTSPGYSPTSPTYSLTSPAISPDDSDEEN (3E8) cumulative living cell number x 6 x 6 8 x 6 6 x 6 x 6 x 6 3 time [days] (E) (G9) (3D) (6D7) viability [%] 9 9 8 8 7 Cell Viability (POL3.3) (3F) 3 time [days] Figure S

YSP T SPS YSP T SPS YSP T SPS 3 3 3 3 3 3 3 6 7 3 6 7 3 6 7 YSTp YST Yp ov Figure S

A P D 7 P Total counts human yeast 7 7 7 OD6 Growth curves yeast CTD variants 6 wt CTD_S_short Yst CTD_S_short Yst 8 6 6 8 6 7 time [min] 3 6 7 B Total counts Human: 6 Y S T S S7 3 Tyrosine -P Yeast: 3 Tyrosine -P E Rpb 8 kd Pol II elution Pol II control (. µg) Boiled beads IgG TEV S P S Y S P T S P S Y S P T S P S Y S P T S P S Y S P T S P S Y S P T S P S Y S P T Threonine -P Threonine -P 8 6 Y S P T S P S Y S P T S P S Y S P T S P S Serine 7-P Y S P T S P S Y S P T S P S Y S P T S P S Serine 7-P F Yst Y S P T S P S Yst Y S P T S P S 3 3 T S P S Y S P T S P S7 Y S P T S P S Y S P T S P S Y S P T S P S7 Y S P T S P S Y S P C Yst Yst EAPTSPG FGVSSPG 3 FSPTSPT YSPTSPA 6 7 8 9 3 6 7 8 9 YSPTSPA 3 YSPTSPN 6 YSPTSPG 7 YSPGSPA 8 YSPKQDEQKHNENENSR 3 6 7 8 9 3 6 7 8 9 3 6 7 8 EAPTSPG FGVSSPG FSPTSPR YSPTSPA -3 YTPTSPR -6 7- - YAPTSPS 3-6-7 8- - YAPTSPK -8 YAPTSPS YSPTSPG YSPGSPA YSPKQDEQKHNENENSR Predicted peptides: EAPTSPGFGVSSPGFSPTSPR YSPTSPA YTPTSPR YAPTSPS YAPTSPK YAPTSPS YSPTSPGYSPGSPAYSPK 3 6 7 8 9 3 6 7 8 9 3 6 7 8 EAPTSPG FGVSSPK FSPTSPT YSPTSPA YAPTSPK - YAPTSPS 3- -6 7-9 YAPTSPS - - -6 7-9 YTPTSPS - YSPTSPN 3- -6 YAPTSPA 7-8 YSPTTPS YSPTTPT YTPTSPK YSPTSPN YSPGSPA YSPKQDEQKHNENENSR Predicted peptides: EAPTSPGFGVSSPK FSPTSPT YSPTSPAYAPTSPK YAPTSPS YAPTSPS YTPTSPSYSPTSPN YAPTSPAYSPTTPS YSPTTPTYTPTSPK YSPTSPN YSPGSPAYSPK 3 6 7 YSTp YST 3 6 7 Yp excluded Figure S3

A 3 FL (µ) - h: DSO. 3 6 FL (µ) - h: DSO. 3 6 FL (µ) - h: DSO. 3 6 kd -Ser-P kd -Ser-P kd -Ser-P -Ser7-P -Ser7-P -Ser7-P -Ser-P -Ser-P -Ser-P -HA -HA B _C _FL _C _FL _C3 _FL3 _C _FL kd Figure S

Supplementary Figures Figure S, related to Figure. (A) (Left) Sequence of mammalian -CTD. Blue letters: non-consensus residues. (Right) List of tryptic CTD peptides comprising CTD repeats 3-. Numbers indicate CTD repeats covered by the corresponding peptide. Peptides 39 and as well as peptides and 8-9 have identical amino acid compositions. (B) List of tryptic CTD peptides of CTD variants,, and. Red letters: amino acid substitution or addition. Blue letters: non-consensus residues. Numbers indicate CTD repeats covered by the corresponding peptide. (C) Western blot analysis showing the stable expression of the five mammalian CTD variants, -, after α-amanitin treatment for weeks, using α-ha (3F/Roche) for detection of the two main Rpb-forms, and. No α-ha signal was detected in -Raji cells (). : marker. (D) Comparison of growth rate (top) and cell viability (bottom) between and CTD variant, which carries the highest number of mutations of all five newly established CTD variants in this study. Daily measurements were performed within a time window of five days. (E) Western blot analysis of rec,, 3 and (endogenous Pol II) using specific antibodies against the various CTD phospho-residues (α-ser-p, Ser-P, Ser7-P, Tyr-P and Thr- P) and total Rpb (α-rpb and α-ha). and designate the hyperphosphorylated and hypophosphorylated forms of the large subunit Rpb of Pol II. α-tubulin was used as a loading control.

Figure S, related to Figure. Scheme of mapped phosphosites within the CTD variants, and. CTD residues are shown by squares. Red: identified phosphoresidue; blue: residue covered in analysis; grey: non p-acceptor residue; black: excluded phosphoresidue due to false positives; yellow: residue not covered in analysis. Figure S3, related to Figure 3. (A) (Left) Total counts of the five different CTD phosphosites (P) within mono-heptads in human (blue) and yeast (red). (Right) Total counts of all possible double-phosphosite combinations (P) within mono-heptads in human (blue) and yeast (red). All repeats with the sequence YSPTSPX (X = S, T, K, R, N, E or G) were included in the final data set. (B) Next neighbor phosphorylation study of Y -P, T -P and S 7 -P in human (Left) and yeast (Right). Only double phosphorylated CTD peptides were taken into account and the defined sequence area comprised - to + CTD residues. Y -P and S 7 -P are underrepresented due to data exclusion from Y -Pfalse positive biased peptides and high number of S 7 replacements within the CTD variants, respectively. (C) CTD sequences of yeast (Left) and yeast CTD variants Yst and Yst are shown. Tryptic peptides of corresponding CTD variants Yst and Yst are listed. Red letters: amino acid substitution. Blue letters: non-consensus residues. Numbers indicate CTD repeats covered by the corresponding peptide. (D) Comparison of growth rates between yeast and yeast CTD variants, Yst and Yst. easurements were performed within a time window of,6 minutes.

(E) Coomassie stained PAA-gel of TAP-purified yeast Rpb of CTD variant Yst after elution. From left to right: arker; Yst-Rpb eluat; Rpb-mass control (. µg); boiled beads only (control); IgG-control; TEV-control; : marker. (F) Scheme of mapped phosphosites within the two yeast CTD variants Yst and Yst. CTD residues are shown by squares. Red: identified phosphoresidue; blue: residue covered in analysis; grey: non p-acceptor residue; black: excluded phosphoresidue due to false positives; yellow: residue not covered in analysis. Figure S, related to Figure. (A) Western blot analysis of Ser-P, Ser-P and Ser7-P after treatment of CTD variant, 3 and with µ flavopiridol (FL) at different time points (h, h,.h, 3h, h and 6h). In addition a DSO control was added and total Rpb-protein was detected by either α-ha ( and 3) or α- Rpb (). α-tubulin was used as a loading control. : marker. (B) Coomassie stained PAA-gel. Four replicates of both, purified -untreated (_C-) and -flavopiridoltreated (_FL-/.h / µ) samples are shown in alternate order from the left to the right. : marker.

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