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.

Figure 1.

Distribution of R-loops in the genome of bloodstream form T. brucei. (A) Analysis of R-loop locations in WT and Tbrh1–/– cells showing DRIP enriched region data sets (middle and right charts) relative to the sequence composition of the 11 T. brucei 11 megabase chromsomes (left chart); genomic elements are colour-coded according to right-hand key. (B) Comparison of the number of WT and Tbrh1–/– DRIP enriched regions and predicted R-loop forming sequences. (C) Screenshot of an RNA Pol II transcribed region of chromosome 1. WT and Tbrh1–/– DRIP data is shown in pink and green, respectively (1–3 on y-axes denotes level of enrichment in DRIP relative to input), with identified enriched regions from each data set shown below, and predicted R-loop forming regions in orange. CDS are shown as thick black lines, UTRs as thin black lines and arrows show direction of transcription; size is indicated. Genes annotated as ‘hypothetical, unlikely’ are shown in grey.

Figure 2.

R-loops are enriched at T. brucei centromeres. (A) Metaplot of DRIP signal in WT (pink) and Tbrh1–/– cell (green) data sets centred on the annotated centromeric interspersed repeats (CIR) ±1 kb. (B) Representative screenshot of a portion of chromosome 2 (Tb927_02_v5.1) containing the centromere region; CDS and DRIP-seq enrichment annotations are shown as in Figure 1, CIR are shown in red.

Figure 3.

DRIP-seq signal at RNA Pol I and Pol III transcribed sites in T. brucei. (A) Graph showing the number of enriched regions identified per kb at Pol I transcribed loci for WT and Tbrh1–/– data sets. (B) A representative screenshot of a rRNA locus is shown, along with histone H3 ChIP-seq data (Wedel et al. 2017) (right; annotation as in Figure 1C). (C) Graph showing DRIP-qPCR targetting three regions of the rRNA locus; RNA Pol I promoter, 5.8S coding region and 28.S coding region. Pecentage of input sequence also detected in IP samples is shown for WT (pink) and Tbrh1–/– (green) cells. In each case EcRHI treated controls are shown as lined bars. Error bars shown SEM of two indepentent replicates. (D) Graph showing the number of enriched regions identified at Pol III transcribed loci. (E) A representative screenshot of a locus containing snRNA and tRNA genes (annotation as in Figure 1C). (F) DRIP-qPCR as in C) targetting two tRNA and two snRNA genes. Error bars show SEM of two independent replicates.

Figure 4.

R-loops within RNA Pol II polycistronic transcription units are predominantly intergenic. (A) Analysis of R-loop locations in WT and Tbrh1–/– cells showing DRIP enriched region data sets within the Pol II PTUs (lower charts) relative to the sequence composition of Pol II PTUs (upper chart); regions are annotated according to the key. (B) DRIP-qPCR, as in Figure 3E, targeting the CDS of six RNA Pol II transcribed genes: NEK22 (Tb927.2.2120), HDAC3 (Tb927.2.2190), ATR (Tb927.11.14680), GPI-8 (Tb927.10.13860), ORC1B (Tb927.9.2030) and actin. (C) Three motifs identified by MEME analysis of WT enriched regions localized to within the Pol II PTUs.

Figure 5.

R-loop accumulation in RNA Pol II transcription units is strongly associated with polyadenylation sites. (A) Metaplots and heatmaps of WT and Tbrh1–/– DRIP signal over the CDS ±1 kb of each Pol II transcribed gene. (B) Metaplots of the number of UTR- or intergenic-associated DRIP enriched regions (red) and PAS (blue) regions per bp for each Pol II CDS ±5 kb for WT (upper) and Tbrh1–/– (lower) DRIP-seq data. (C) Prepresentative screenshot of WT DRIP signal (pink) relative to mapped PAS (blue; predominantly used PAS in dark blue) and SAS (orange) locations in a region of chromosome 5; CDS and DRIP-seq enrichment annotations are as shown as in Figure 1.

Figure 6.

R-loop accumulation is the mirror of nucleosome accumulation and is dictated by RNaseH1 throughout RNA Pol II transcription units. (A) Metaplots of WT (pink) and Tbrh1–/– (green) DRIP signal over the ATG (±500 bp) of the first gene of each Pol II PTU (left) and over all other genes in the PTU (right). (B) Metaplot analysis of DRIP (blue) and histone H3 ChIP (red) signal over the ATG (±500 bp) of the first gene of each Pol II PTU (left) and all other Pol II transcibed genes (right).

Figure 7.

R-loop distribution is equivalent at strand switch regions at which replication initiation has been mapped, or where replication initiation has not been detected. (A) Metaplot analysis of WT (pink) and Tbrh1–/– (green) DRIP signal over replication origin (ORI)-associated SSRs (left), with a representative screenshot of one such SSR (right; gene and DRIP-seq enrichment annotations are as shown in Figure 1). (B) Metaplot (left) and representative screenshot (right) of SSRs where no replication origin (no ORI) activity has been detected.

Figure 8.

R-loop enrichment shows a greater association with multigenic transcripton initiation and associated markers than termination.The upper diagrams show metaplot profiles of WT (pink) and Tbrh1–/– (green) DRIP signal over divergent (A), head-to-tail (B) and convergent (C) SSRs (±1 kb), and the middle diagrams are representative screenshots of each (gene and DRIP-seq enrichment annotations are as shown in Figure 1, and locations of tRNA genes as red boxes). In the lower diagrams the WT DRIP signal (blue) metaplot is compared to H2A.Z ChIP signal (62) over divergent (left), head-to-tail (middle) and convergent (right) SSRs (±8 kb).

Figure 9.

A summary of R-loop localization at mRNA processing regions in T. brucei multigenic RNA Pol II transcription units. Genes within Pol II PTUs are separated by UTR and intergenic DNA sequences containing sites of polyadenylation (poly(A)) and splice acceptor addtion (AG, where the splice leader (SL) 5′ cap is trans spliced), which are known to be directed by polypyrimidine (poly(Y)) tracts that are critical for correct maturation of the mRNA. R-loops containing RNA (red) putatively emerging from RNA Pol II (blue) and hybridizing to the DNA (black)form over these regions between the CDS of adjacent genes, within areas of nucleosome (grey) depletion, and are acted upon by T. brucei RNaseH1 (green).