Recruitment of P-TEFb (Cdk9-Pch1) to chromatin by the cap-methyl transferase Pcm1 in fission yeast.
Capping of nascent pre-mRNAs is thought to be a prerequisite for productive elongation and associated serine 2 phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (PolII). The mechanism mediating this link is unknown, but is likely to include the capping machinery and P-TEPb. We report that the fission yeast P-TEFb (Cdk9-Pch1) forms a complex with the cap-methyltransferase Pcm1 and these proteins colocalise on chromatin. Ablation of Cdk9 function through chemical genetics causes growth arrest and abolishes serine 2 phosphorylation on the PolII CTD. Strikingly, depletion of Pcm1 also leads to a dramatic decrease of phospho-serine 2. Chromatin immunoprecipitations show a severe decrease of chromatin-bound Cdk9-Pch1 when Pcm1 is depleted. On the contrary, Cdk9 is not required for association of Pcm1 with chromatin. Furthermore, compromising Cdk9 activity leads to a promoter-proximal PolII stalling and sensitivity to 6-azauracil, reflecting elongation defects. The in vivo data presented here strongly support the existence of a molecular mechanism where the cap-methyltransferase recruits P-TEFb to chromatin, thereby ensuring that only properly capped transcripts are elongated.
Guiguen
Allan
A
Laboratoire de Génétique Moléculaire (GEMO), Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium.
Soutourina
Julie
J
Dewez
Monique
M
Tafforeau
Lionel
L
Dieu
Marc
M
Raes
Martine
M
Vandenhaute
Jean
J
Werner
Michel
M
Hermand
Damien
D
D015153:Blotting, Western; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D047468:Immunoprecipitation; D008780:Methyltransferases / Q000378:metabolism*; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012568:Schizosaccharomyces; D014158:Transcription, Genetic / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D014498:Uracil / Q000031:analogs & derivatives
D002843:Chromatin; D014498:Uracil; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D042925:Positive Transcriptional Elongation Factor B; D012319:RNA Polymerase II; C100248:azauracil