Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids

Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E.; Pandya, Unnati M.; Li, Bibo

doi:10.1093/nar/gkx184

Cited by 77 publications

(164 citation statements)

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“…Nonetheless, R-loop association with apparently all T. brucei centromeres appears to reveal a conserved function for these DNA elements, perhaps related to chromatin modification (117) or cell cycle signalling (118), despite pronounced divergence of the kinetoplastid kinetochore complex that binds centromeres (119,120). Finally, previous work in T. brucei (121) and Leishmania (122) has revealed the existence of TERRA RNA, with over expression of TbRH1 suppressing the levels of R-loops at T. brucei telomere repeats. However, the extent to which (sub)telomeric R-loops might contribute to kinetoplastid biology, such as the expression or stability of telomere-proximal genes, has been little explored.…”

Section: Discussionmentioning

confidence: 81%

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

Briggs

Hamilton

Crouch

et al. 2018

Preprint

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R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere homeostasis, 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 transsplicing 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 localisation 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 intergenic regions of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination, most T. brucei R-loops act in a novel role, promoting RNA Pol II movement or mRNA processing. Finally, we show there is little evidence for correlation between R-loop localisation and mapped sites of DNA replication initiation.

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Section: Discussionmentioning

confidence: 81%

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

Briggs

Hamilton

Crouch

et al. 2018

Preprint

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“…In WT cells there was limited read enrichment across the ES region spanning the promoter to the VSG , either in the actively transcribed ES (BES1, containing VSG221 ) or the 13 distinct silent ES. Pronounced enrichment was only observed proximal to the ends of the ES, downstream of the VSG, which most likely represents TERRA RNA since levels of the signal increased in Tbrh1 -/- mutants (Figs.2A,E), the opposite of decreased TERRA RNA when TbRH1 is over-expressed (26). Loss of TbRH1 resulted in DRIP-seq signal throughout all ES, both active and silent (Fig.2A, Fig.S3).…”

Section: Resultsmentioning

confidence: 99%

RibonucleaseH1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion

Briggs

Crouch

Lemgruber

et al. 2018

Preprint

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30Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune 31 evasion strategy that is catalysed both by transcription and recombination reactions, each operating within 32 specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the 33 single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here 34 we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp 35 repeats in the actively transcribed ES, but spread throughout the active and inactive ES in the absence of 36 RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching 37 and replication-associated genome damage, some of which accumulates within the active ES. This work 38indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei 39 immune evasion by transcription and recombination. 40 41 Author summary 42All pathogens must survive eradication by the host immune response in order to continue infections and be 43 passed on to a new host. Changes in the proteins expressed on the surface of the pathogen, or on the 44 surface of the cells the pathogen infects, is a widely used strategy to escape immune elimination. 45Understanding how this survival strategy, termed antigenic variation, operates in any pathogen is critical, 46 both to understand interaction between the pathogen and host and disease progression. A key event in 47 antigenic variation is the initiation of the change in expression of the surface protein gene, though how this 48 occurs has been detailed in very few pathogens. Here we examine how changes in expression of the surface 49 coat of the African trypanosome, which causes sleeping sickness disease, are initiated. We reveal that 50 specialised nucleic acid structures, termed R-loops, form around the expressed trypanosome surface 51 protein gene and increase in abundance after mutation of an enzyme that removes them, leading to 52 increased changes in the surface coat in trypanosome cells that are dividing. We therefore shed light on the 53 earliest acting events in trypanosome antigenic variation. 54 55 56 Introduction 57

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“…RAP1 knockdown results not only in ES depression but also in an increase in double-strand DNA breaks at the ES telomere, with a concurrent increase in VSG switching mediated by DNA recombination [41]. Intriguingly, this appears to occur through transcriptional regulation of a non-coding RNA.…”

Section: How Is Vsg Expression Controlled In a Monoallelic Fashion?mentioning

confidence: 99%

How to create coats for all seasons: elucidating antigenic variation in African trypanosomes

Ooi

Rudenko

2017

Emerging Topics in Life Sciences

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Extracellular parasites of the mammalian bloodstream face considerable challenges including incessant assault by the immune system. African trypanosomes are consummate survivors in this inclement environment and are renowned for their supremely sophisticated strategy of antigenic variation of their protective surface coat during the course of chronic infections. Recent developments are making us realize how complex this antigenic machinery is and are allowing us to tackle previously intractable problems. However, many of the simplest (and arguably the most important) questions still remain unanswered! How is the extraordinary heterogeneity of variant surface glycoprotein variants during a chronic infection generated?The bloodstream form of Trypanosoma brucei is coated with a dense covering of ∼10 7 variant surface glycoprotein (VSG) molecules, which extend as antigenically variable rods connected to the cell surface via glycosylphosphatidylinositol (GPI) anchors [1]. This GPI attachment allows VSGs to move freely over the cell surface [2]. Tight packing of these VSGs produces a protective barrier shielding invariant surface proteins from recognition by host antibodies and preventing trypanosome lysis by the alternative pathway of the complement system [3]. Extraordinarily high rates of recycling of surface VSG allow selective removal of host cell molecules including antibodies and complement from the trypanosome surface, thereby functioning as a 'coat-cleaning ' machine [4]. This prolongs trypanosome survival in rising antibody titres and facilitates escape from macrophages [5].Each trypanosome has a vast repertoire of VSG genes [6], differing in number and content between different strains, presumably as a consequence of diversifying forces. For example, the 'VSGnome' of T. brucei 427 contains more than 2500 different VSG genes, of which more than 80% are not immediately functional [7]. Switching the active VSG can entail transcriptional switches between the ∼15 telomeric VSG expression site (ES) transcription units (Figure 1). More importantly, DNA rearrangements and predominantly gene conversions can copy new VSGs (or segments of VSGs) into the active ES, thereby resulting in an antigenic switch [8] (Figure 1). This extraordinary ability to construct novel patchwork, or mosaic, VSGs is key for trypanosome survival [9]. The continuous recombination of various permutations and combinations of sections of VSG genes (or pseudogenes) allows the trypanosome to construct an almost infinite number of antigenically distinct VSG coat types.A given wave of trypanosome parasitaemia is normally predominantly composed of a major VSG variant, although it has long been known that minor VSG variants are also present within each wave. However, there is possibly phenomenal heterogeneity within these infection waves, as next-generation RNA sequencing of RNA transcripts has documented an extraordinary variety of minor VSG variants [10]. These experiments argue that a considerable percentage of the available VSG repertoire is disp...

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Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids

Cited by 77 publications

References 50 publications

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

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

RibonucleaseH1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion

How to create coats for all seasons: elucidating antigenic variation in African trypanosomes

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