The export factor Mex67 sets boundaries for polymerase I transcription in Trypanosoma brucei

POZZI, BERTA; NAGULESWARAN, ARUNASALAM; RODITI, ISABEL

Woods Hole

Congreso; XXXII Molecular Parasitology Meeting; 2021

Consistent with the established fact of polycistronic transcription in trypanosomes, ChIP-seq data from our laboratory did not show striking differences in Pol II occupancy across polycistronic transcription units (PTU) [1]. By contrast, while global run-on sequencing (GRO-Seq) gives fairly homogeneous incorporation of 5-ethynyl-uridine (5EU) in some PTUs, we find that others show marked differences between adjacent genes. To explore the mechanisms underlying this uneven incorporation, and since co-transcriptional mRNA export in T. brucei has been reported recently [2], we performed GRO-seq with cells depleted of export factors by RNAi. Although we did not find significant differences in overall 5EU incorporation, two regions of the genome on chromosomes 6 and 10 showed heavy labelling upon depletion of Mex67 and complementation with a dominant negative variant Mex67-DN. These regions span the procyclin loci which, in contrast to most protein-coding genes, are transcribed by RNA Pol I. 5EU labelling and Pol I ChIP- seq suggest that readthrough transcription proceeds until Pol II transcription start sites on the opposite strand, easily identifiable by a histone H3K4me3 trimethylation mark. Mex67-DN lacks the N-terminal zinc finger (nucleotide binding) motif, only present in Trypanosomatids [3], and seems to have a reduced nuclear localisation compared to wild-type Mex67. This might explain its diminished binding to DNA at the procyclin loci and other highly transcribed regions, as determined by ChIP-seq. These findings, together with its interaction with RNA Pol II and the chromatin remodelling factor RRM1, support a novel role for Mex67 in regulating transcription in Trypanosomatids. [1] PMID: 30544263 [2] PMID: 30418648 [3] PMID: 23560737