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

Abstract

ABSTRACT Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 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 m 7 G capping enzymes. The first critical step, Ser 5 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 parasite Trypanosoma brucei , however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. 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 m 7 G capping. Instead, we observed that CRK9 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.