Pozzi, Maria BertaMaria BertaPozziNaguleswaran, ArunasalamArunasalamNaguleswaran0000-0002-9509-485XFlorini, FrancescaFrancescaFlorini0000-0003-2579-3820Rezaei, ZahraZahraRezaeiRoditi, IsabelIsabelRoditi0000-0003-2812-65132024-10-252024-10-252023-06-09https://boris-portal.unibe.ch/handle/20.500.12422/166558TbMex67 is the major mRNA export factor known to date in trypanosomes, forming part of the docking platform within the nuclear pore. To explore its role in co-transcriptional mRNA export, recently reported in Trypanosoma brucei, pulse labelling of nascent RNAs with 5-ethynyl uridine (5-EU) was performed with cells depleted of TbMex67 and complemented with a dominant-negative mutant (TbMex67-DN). RNA polymerase (Pol) II transcription was unaffected, but the procyclin loci, which encode mRNAs transcribed by Pol I from internal sites on chromosomes 6 and 10, showed increased levels of 5-EU incorporation. This was due to 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. Complementation by TbMex67-DN also increased Pol I-dependent formation of R-loops and γ-histone 2A foci. The DN mutant exhibited reduced nuclear localisation and binding to chromatin compared to wild-type TbMex67. Together with its interaction with chromatin remodelling factor TbRRM1 and Pol II, and transcription-dependent association of Pol II with nucleoporins, our findings support a role for TbMex67 in connecting transcription and export in T. brucei. In addition, TbMex67 stalls readthrough by Pol I in specific contexts, thereby limiting R-loop formation and replication stress.en500 - Science::570 - Life sciences; biologyarticle10.48350/1818383707019610.1093/nar/gkad251