Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr1 -Ser2 -Pro3 -Thr4 -Ser5 -Pro6 -Ser7 in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m7 G capping enzymes. The first critical step, Ser5 phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasiteTrypanosoma brucei , however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. TheT. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression ofT. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m7 G capping. Instead, we observed thatCRK9 silencing led to a block of spliced leader (SL)trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.