title: Trypanosome cdc2-related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of RNA polymerase II subunit RPB1.
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.
authors.affiliation: Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Nitika
authors.initials: N
authors.lastname: Badjatia
authors.forename: Daniela L
authors.initials: DL
authors.lastname: Ambrósio
authors.forename: Ju Huck
authors.initials: JH
authors.lastname: Lee
authors.forename: Arthur
authors.initials: A
authors.lastname: Günzl
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D008745:Methylation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D020038:RNA, Spliced Leader / Q000378:metabolism; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D021122:Protein Subunits; D015800:Protozoan Proteins; D012315:RNA Caps; D020038:RNA, Spliced Leader; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II