Exploring the relevance of co-transcriptional export and degradation in Trypanosoma brucei

POZZI, BERTA; NAGULESWARAN, ARUNASALAM; RODITI, ISABEL

Heidelberg

Conferencia; 14th EMBL Comference on Transcription and Chromatin; 2020

EMBL

It is almost an article of faith that there is no transcriptional control of Pol II in trypanosomes. Consistent with this, across a polycistronic transcription unit, ChIP-seq data from our lab did not show striking differences in Pol II occupancy while GRO-Seq studies from Tryps pulsed with 5EU showed there are regions of the genome with fairly homogeneous amount of reads. However, other regions had marked differences in incorporation between adjacent genes that were also in the same polycistronic transcription unit. Heavily labelled transcripts frequently encoded ribosomal proteins and mitochondrial components. In an attempt to reveal the underlying mechanism responsible for this uneven incorporstion, and since co-transcriptional export in Tryps has been reported recently, we performed GRO-seq from either pulsed live cells or isolated nuclei and also from cells in which different export factors had been knocked-down. Strikingly, we did not find significant differences in overall 5EU incoporation, suggesting that export is not the main responsible for the pattern observed. Nevertheless, some regions of the genome, particularly in Chromosomes VI and X, showed profusive labelling downstream of procyclins loci upon depletion of the export factor Mex67 and complementation with a dominant negative variant that lacks it N-terminal zinc finger domain. In this case, 5EU labelling suggest that profusive transcription proceeds until PolII tanscription initiation sites on the opposite strand, easily identifiable by H3K4 methylation mark. Since ChIP experiments revealed that both PolI and PolII are actually in those loci, we propose to elucidate whether these antisense transcripts are the result of deregulated transcription or accumulation upon export blockage. The former is further supported by the fact that Mex67 not only harbours a zinc finger domain but also was found in PolII transcription complex. To asses the later, we will perform GRO-seq from exosome components RNAi cell lines.