Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes

Abstract

Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.

Mex67 paralogs mediate division of labor in trypanosome RNA processing and export

“…Genes are transcribed, capped, introns spliced out, polyadenylated and export occurs co-transcriptionally, with the basket playing a prominent role in quality control prior to mRNA export. Trypanosomes have histone marks and single sequence specific promoters for each polycistronic transcription unit as opposed to individual gene promoters (Berriman et al, 2005; Cordon-Obras et al, 2022; Siegel et al, 2009). Transcription is polycistronic with individual mRNA resolved by trans splicing of a splice leader sequence common to every single mRNA and then poly adenylated (comprehensively reviewed in (Kramer, 2021)).…”

“…However, there is a significant enrichment of mRNA encoding RNA binding proteins (RBPs) on TbMex67 as compared with TbMex67b, suggesting a role in control of gene expression given the role of RBPs in the post transcriptional regulation of genes in trypanosomes (Clayton, 2013). (Berriman et al, 2005;Cordon-Obras et al, 2022;Siegel et al, 2009). Transcription is polycistronic with individual mRNA resolved by trans splicing of a splice leader sequence common to every single mRNA and then poly adenylated (comprehensively reviewed in (Kramer, 2021)) .…”

“…Of these, trypanosomes represent both an extremely distant relative of animals/fungi and significant work has demonstrated very unusual transcriptional mechanisms. Trypanosomes lack canonical RNA polymerase II (Pol II) promoters for individual genes, instead relying on histone modifications and sequence specific promoters to drive polycistronic transcription of multiple genes with resolution to single mRNAs by trans -splicing and polyadenylation (Berriman et al, 2005; Cordon-Obras et al, 2022; Siegel et al, 2009). While mRNA stability is a key component of gene regulation in trypanosomes (Clayton, 2013), the role of mRNA export in regulating mRNA transcripts remains poorly characterized.…”

“…Genes are transcribed, capped, introns spliced out, polyadenylated and export occurs co-transcriptionally, with the basket playing a prominent role in quality control prior to mRNA export. Trypanosomes have histone marks and single sequence specific promoters for each polycistronic transcription unit as opposed to individual gene promoters(Berriman et al, 2005;Cordon-Obras et al, 2022;…”

Deciphering divergent trypanosomatid nuclear complexes by analyzing interactomic datasets with AlphaFold2 and genetic approaches

“…Some promoters have been identified for genes like the SL RNA locus, rRNA locus, procyclin and VSG genes of T. brucei 6 . Recently, a promoter sequence was reported in the 5’ region of gene tandem units in T. brucei , but the significance of this finding remains yet-to-be well determined 7 . They also contain a set of basal transcription factors that form a very divergent initiation complex 8 .…”