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 (CRK9AS). Inhibition of CRK9AS 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.

The pre-mRNA splicing factor PRP19 is recruited into the spliceosome after forming the PRP19/CDC5L complex in humans and the Nineteen complex in yeast. Additionally, ‘PRP19-related’ proteins enter the spliceosome individually or in pre-assemblies that differ in these systems. The protistan family Trypanosomatidae, which harbors parasites such as Trypanosoma brucei, diverged early during evolution from opisthokonts. While introns are rare in these organisms, spliced leader trans splicing is an obligatory step in mRNA maturation. So far, ∼70 proteins have been identified as homologs of human and yeast splicing factors. Moreover, few proteins of unknown function have recurrently co-purified with splicing proteins. Here we silenced the gene of one of these proteins, termed PRC5, and found it to be essential for cell viability and pre-mRNA splicing. Purification of PRC5 combined with sucrose gradient sedimentation revealed a complex of PRC5 with a second trypanosomatid-specific protein, PRC3, and PRP19-related proteins SYF1, SYF3 and ISY1, which we named PRP19-related complex (PRC). Importantly, PRC and the previously described PRP19 complex are distinct from each other because PRC, unlike PRP19, co-precipitates U4 snRNA, which indicates that PRC enters the spliceosome prior to PRP19 and uncovers a unique pre-organization of these proteins in trypanosomes.

Known transcription factors of trypanosomatid organisms are extremely divergent in amino acid sequence to their counterparts in other eukaryotes. Sequence similarity is so limited that factors have been primarily identified by functional and structural studies. In addition, trypanosomatids may have evolved factors that are specific to this group of organisms. Under these circumstances, an in vitro transcription system is invaluable as it allows for unambiguous determination of a factor's transcriptional role. Here we describe procedures for the preparation of transcriptionally active extracts, detail in vitro transcription reactions, and specify the particular strategy necessary to detect template-derived RNA in this system. As examples of how to use this system, we describe factor depletion from extract and antibody-mediated interference with a factor's transcriptional function. Furthermore, we detail a promoter pull-down assay that makes use of the extracts and facilitates analysis of a factor's interaction with promoter DNA.

Ciliary stability and function are regulated by the covalent addition of polyglutamate and polyglycine chains to axonemal tubulin subunits. The Drosophila gene CG13442 encodes a predicted ubiquitin E3 ligase involved in the regulation of tubulin glycylation and glutamylation. Homologous to mammalian MARCH8, CG13442/dMARCH8 is required for male fertility. Sperm in dMARCH8 mutant testes appear to undergo a normal individualization process but fail to be transferred to the seminal vesicle. This phenotype is very similar to that of mutants in the Ntl gene, shown here to be a glycine transporter using a [3H]glycine uptake assay. Mutations in dMARCH8 are associated with a reduction of both polyglutamylation and polyglycylation of sperm tubulin. Polyglutamylation of tubulin is significantly increased in the Ntl− background, and recovers to wild-type levels in the Ntl−-dMARCH8− double mutant background, indicating that glycine and glutamate compete for some common site(s) on tubulin molecules in this system. By analogy to the regulation of the mammalian glycine transporter GlyT2 through ubiquitin-mediated trafficking between the plasma membrane and endosome, dMARCH8 may target Ntl and glutamate transporters, or other upstream regulators of these proteins.