The nuclear proteome of Trypanosoma brucei

Abstract

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes.

Discovery of new mitochondrial DNA segregation machinery components inTrypanosoma brucei: a comparison of three approaches

“…Two of the three high confidence p166 clones expressed a short region from amino acid 37 to 210 while the third clone expressed a larger fragment ranging from amino acid 71 to 716 of the 1502 amino acid long protein ( Figure S4). The only other hits with good confidence were two identical clones of the C-terminal region of the putative nuclear pore component NUP109, that was previously found in a proteomics analysis characterising nuclear proteins (Goos et al 2017) .…”

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

“…PP1-1 has the highest sequence homology to the PP1 involved in termination in yeast and humans [54]. PP1-7 has been identified in the nucleus of T. brucei [58]. However, neither of these PP1 proteins associates with TbPNUTS in co-IP experiments (S3C Fig).…”

“…In contrast to Leishmania, base J synthesis is developmentally regulated in T. brucei, where synthesis is lost upon differentiation to PCF due to the downregulation of JBP1 and JBP2 [22,38,44,57]. While JGT has been shown to be part of the nuclear proteome in PCF T. brucei [58] and significant level of JGT activity is present in PCF, as shown by the presence of J in parasites grown in the presence of the J precursor hmU [38,44], JGT mRNA is downregu-lated~2-fold between BSF and PCF [59]. Therefore, to fully characterize the association of JGT with the TbPNUTS complex, we need to utilize BSF T. brucei.…”

A DOT1B/Ribonuclease H2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation inTrypanosoma brucei

“…As DOT1B has a nuclear localization (73), and acts as a histone methyltransferase on chromatin (56), we further focused on proteins predicted to be nuclear. Four proteins, besides DOT1B were categorized as such based on nuclear proteome data (74), TrypTag (73), and relevant literature (as indicated in Supplementary Table S1).…”