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.
Highlights
RNA–DNA hybrids display enhanced stability compared with double-stranded DNA or RNA due to the unusual conformation they adopt [1,2]
We show that R-loops are very abundant across the T. brucei genome, with the most pronounced genomic localization seen throughout the RNA Pol II multigene transcription units, where R-loops coincide most clearly with polyadenylation sites and regions of nucleosome depletion
DRIP-seq mapping to the T. brucei genome revealed very widespread coverage (Figure 1): based on >1.2-fold enrichment of reads in the DRIP sample relative to input, ∼35 000 enriched regions were predicted in both wild type (WT) and Tbrh1–/– cells
Summary
RNA–DNA hybrids display enhanced stability compared with double-stranded DNA or RNA due to the unusual conformation they adopt [1,2]. Small RNA–DNA hybrids form during priming of DNA replication and within RNA polymerase (Pol) during transcription, whereas larger RNA–DNA hybrids, termed R-loops, can form when RNA exiting the RNA Pol can sometimes access the DNA before the duplex reanneals These R-loops are exacerbated when elements of RNA biogenesis are impaired [3,4,5,6,7] and are increasingly recognized as providing widespread roles [8,9,10], which are not all co-transcriptional. R-loops may form in trans, when RNA moves from the site of its genesis to another, homologous location. Eukaryotic and bacterial homologous recombination proteins (which normally direct DNA repair) are capable of generating RNA–DNA hybrids [12,13,14,15]
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