Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

Abstract

R-loops are stable RNA–DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere maintenance, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localization was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the regions between coding sequences of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination the most widespread localization of R-loops in T. brucei suggests a novel correlation with pre-mRNA processing. Finally, we find little evidence for correlation between R-loop localization and mapped sites of DNA replication initiation.

Trypanosoma bruceiribonuclease H2A is an essential enzyme that resolves R-loops associated with transcription initiation and antigenic variation

“…Correspondingly, the number of DRIP enriched regions associated with gene coding DNA sequence (CDS) was reduced in both the TbRH2A uninduced (6,715 regions) and, even more so, in the RNAi induced (5,300 regions) DRIP-seq data compared with WT (8861 regions; Fig.S6A). Heatmaps of DRIP-seq enrichment around every RNA Pol II gene confirmed the predominant enrichment around the CDS, with relatively precise signal localisation upstream and downstream of each CDS, as was seen in WT cells (Fig.S7) and TbRH1 null mutants [47]. Taken together, these data indicate relatively stable accumulation of R-loops within the RNA Pol II PTUs, which is not markedly altered by loss of TbRH2A or TbRH1 [47].…”

“…Fig.S4 shows genome-wide DRIP-seq mapping after 24 hr of growth with and without tet-induced RNAi, revealing widespread R-loop enrichment and some correlation with DNA repeats. To understand how R-loop distribution in the TbRH2A RNAi cells compared with similar analysis in TbRH1 null mutants and WT T. brucei cells [47], DRIP-enriched regions were defined as locations with ≥1.2 fold-change increase in IP mapped reads relative to pre-IP samples. Analysis of these enriched regions showed they were mainly found in RNA Pol II PTUs (∼88% of uninduced, and ∼86% of RNAi induced; Fig.S5), a very similar distribution to that previously reported for WT and TbRH1 null mutant DRIP-seq data.…”

“…Transcription in T. brucei also appears functionally linked with DNA replication, since at least one component of the origin recognition complex (ORC) binds to SSRs, although only a subset are activated during S phase [45, 46]. We have shown that the most abundant site of R-loop accumulation in the constitutively transcribed T. brucei ‘core’ genome is at intergenic sequences within the RNA pol II PTUs, where the RNA-DNA hybrids display precise association with regions of low nucleosome density, suggesting a relationship with polyadenylation and, perhaps, trans -splicing [47]. R-loops are also notably enriched at SSR boundaries where RNA Pol II transcription initiates, whereas little or no R-loop signal is observed at SSRs where transcription termination takes place.…”

“…R-loops are also notably enriched at SSR boundaries where RNA Pol II transcription initiates, whereas little or no R-loop signal is observed at SSRs where transcription termination takes place. Although R-loops increase at various loci after deletion of TbRH1 [47], no cell cycle or growth defects are observed, even though it might be predicted that R-loops in the PTUs present an obstacle to replication and transcription, or that they might form at potentially predictable sites of clashes between the T. brucei replication and transcription machineries, perhaps suggesting TbRH1-independent mechanisms to avoid such conflict.…”

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

“…brucei (65). Thus, it seems conceivable that SSRs are also sites of such interaction in Leishmania, though no equivalent mapping of ORC or RNA-DNA hybrids has been reported.…”