The RNA export factor TbMex67 connects transcription and RNA export in Trypanosoma brucei and sets boundaries for RNA polymerase I.

In trypanosomatids, all protein-coding genes transcribed by RNA polymerase II (Pol II) are organised in long polycistronic units. Another unusual feature of Trypanosoma brucei is that its major surface glycoproteins are transcribed by RNA Pol I. We recently established procedures for labelling nascent RNAs with 5-ethynyl uridine (5-EU) for high-throughput sequencing. This revealed that different transcription units and, more surprisingly, genes within the same polycistronic transcription unit, showed vastly different levels of incorporation of 5-EU. To explore whether these differences were due to co-transcriptional export, we performed metabolic labelling of cells depleted of the general RNA export factor TbMex67. We found two regions of the genome showed increased levels of nascent transcription upon depletion of TbMex67 and complementation with a dominant-negative variant (TbMex67-DN). These regions span the procyclin loci, which are transcribed by Pol I. ChIP-seq revealed that this was the result of Pol I readthrough transcription, which proceeded beyond the procyclin and procyclin-associated genes up to the Pol II transcription start site on the opposite strand. Depletion of TbMex67 and complementation by the dominant negative mutant also led to increases in R-loops and DNA damage. TbMex67-DN, which lacks the N-terminal zinc finger (nucleotide binding) motif unique to trypanosomatids, exhibited reduced nuclear localisation compared to wild-type TbMex67 and diminished binding to chromatin at the procyclin loci and other highly transcribed regions. These findings, together with its interaction with the RNA-binding and chromatin-remodelling factor TbRRM1, supports novel roles for TbMex67 in i) connecting export and transcription machineries in T. brucei, ii) preventing readthrough by Pol I in specific contexts and iii) reducing DNA damage by preventing R-loop formation.