Depletion of Trypanosome CTR9 Leads to Gene Expression Defects

Abstract

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1.

Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

“…S6A in the supplemental material). Intriguingly, RNAi targeting CTR9, which might be involved in transcription elongation (57), showed an even stronger effect (Fig. 7C).…”

“…In the second experiment, all copurifying proteins were analyzed. To find out whether proteins were specifically associated with PUF2, we compared the new results with those for other purifications (unpublished results) (57). Numerous proteins copurified specifically with PUF2, but none of them was found in both purifications (see Table S2 in the supplemental material).…”

Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks

“…Some of these interactions are probably specific, but many of the proteins concerned have been found in numerous other purifications (57). Although the purification did not contain RNase inhibitors, some RNA-mediated associations could have persisted.…”

The many roles of the conserved eukaryotic Paf1 complex in regulating transcription, histone modifications, and disease states

“…Identification of RNAs directly dependent on yPaf1 showed an overrepresentation of essential genes, which may explain why paf1 Δ cells have compromised fitness [24]. Very recently, Paf1C homologues have been found to influence expression of many essential genes in the obligate parasite Trypanosoma brucei , which suggests that functions of Paf1C components may be even more broadly conserved then previously appreciated [91]. …”