Rapid block of pre‐mRNA splicing by chemical inhibition of analog‐sensitive CRK9 in Trypanosoma brucei

Trypanosoma brucei CRK9 is an essential cyclin‐dependent kinase for the parasite‐specific mode of pre‐mRNA processing. In trypanosomes, protein coding genes are arranged in directional arrays that are transcribed polycistronically, and individual mRNAs are generated by spliced leader trans ‐splicing and polyadenylation, processes that are functionally linked. Since CRK9 silencing caused a decline of mRNAs, a concomitant increase of unspliced pre‐mRNAs and the disappearance of the trans ‐splicing Y structure intermediate, CRK9 is essential for the first step of splicing. CRK9 depletion also caused a loss of phosphorylation in RPB1, the largest subunit of RNA polymerase (pol) II. Here, we established cell lines that exclusively express analog‐sensitive CRK9 (CRK9 AS ). Inhibition of CRK9 AS in these cells by the ATP‐competitive inhibitor 1‐NM‐PP1 reproduced the splicing defects and proved that it is the CKR9 kinase activity that is required for pre‐mRNA processing. Since defective trans‐ splicing was detected as early as 5 min after inhibitor addition, CRK9 presumably carries out reversible phosphorylation on the pre‐mRNA processing machinery. Loss of RPB1 phosphorylation, however, took 12–24 hr. Surprisingly, RNA pol II‐mediated RNA synthesis in 24 hr‐treated cells was upregulated, indicating that, in contrast to other eukaryotes, RPB1 phosphorylation is not a prerequisite for transcription in trypanosomes.