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Review
. 2020 Jul 10;432(15):4167-4185.
doi: 10.1016/j.jmb.2019.10.025. Epub 2019 Nov 2.

Telomere and Subtelomere R-loops and Antigenic Variation in Trypanosomes

Arpita Saha  1 , Vishal P Nanavaty  1 , Bibo Li  2
Affiliations
Review

Telomere and Subtelomere R-loops and Antigenic Variation in Trypanosomes

Arpita Saha et al. J Mol Biol. .

Abstract

Trypanosoma brucei is a kinetoplastid parasite that causes African trypanosomiasis, which is fatal if left untreated. T. brucei regularly switches its major surface antigen, VSG, to evade the host immune responses. VSGs are exclusively expressed from subtelomeric expression sites (ESs) where VSG genes are flanked by upstream 70 bp repeats and downstream telomeric repeats. The telomere downstream of the active VSG is transcribed into a long-noncoding RNA (TERRA), which forms RNA:DNA hybrids (R-loops) with the telomeric DNA. At an elevated level, telomere R-loops cause more telomeric and subtelomeric double-strand breaks (DSBs) and increase VSG switching rate. In addition, stabilized R-loops are observed at the 70 bp repeats and immediately downstream of ES-linked VSGs in RNase H defective cells, which also have an increased amount of subtelomeric DSBs and more frequent VSG switching. Although subtelomere plasticity is expected to be beneficial to antigenic variation, severe defects in subtelomere integrity and stability increase cell lethality. Therefore, regulation of the telomere and 70 bp repeat R-loop levels is important for the balance between antigenic variation and cell fitness in T. brucei. In addition, the high level of the active ES transcription favors accumulation of R-loops at the telomere and 70 bp repeats, providing an intrinsic mechanism for local DSB formation, which is a strong inducer of VSG switching.

Keywords: 70 bp repeats; Antigenic variation; R-loop; TERRA; Telomere.

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Figures

Figure 1.
Figure 1.
(A) Schematic diagram of a megabase chromosome (top), an intermediate chromosome (middle), and a minichromosome (bottom) in T. brucei, with their type, number, and size indicated. The central region of the two homologous megabase chromosomes are the same and is shown once, while their subtelomeres are different and shown separately (top). Individual genes are represented as short colored bars. Grey, functional genes; red, VSG genes; yellow, pseudogenes. PTUs and their transcription directions are marked with arrows. VSG gene arrays, a B-ES, and an M-ES are shown. A B-ES is shown at one subtelomere of the intermediate chromosome (middle). Individual VSG genes are shown at subtelomeres of the minichromosome (bottom) (B) A representative B-ES (top) and an M-ES (bottom) is shown. ESAGs: Expression Site Associated Genes.
Figure 2.
Figure 2.
(A) VSG switching mechanisms. Light blue soft arrow heads, telomere repeats; purple arrow heads, 70 bp repeats; red and dark blue 3D arrows, VSG genes; green 3D arrows, ESAGs; long red line with an arrow head, active transcription from the B-ES promoter; grey short line with an arrow head, short-distance transcription from the silent B-ES promoter. VSG switching pathways are explained in the text. (B) The classical double Holliday Junction pathway of homologous recombination to generate either GC or CO products is shown on the left. In comparison, the BIR pathway that usually occurs at the telomere is shown on the right. The BIR and GC products are indistinguishable because the telomeres downstream of the VSG genes have the same tandem repeat sequence.
Figure 3.
Figure 3.
(A) Subnuclear localization of TERRA. Top, TERRA FISH was performed in BF WT T. brucei cells with an Alexa488-conjugated PNA probe containing (CCCTAA)3 (TELC probe, PNA Bio). Hybridization was done at 37°C without denaturation. Treating cells with RNase A eliminated all nuclear punctate signals (right image in the top row), confirming that the observed signals represent RNA molecules. Bottom, TbTRF IF was performed using a rabbit antibody recognizing TbTRF and an Alexa594-conjugated donkey anti-rabbit 2nd antibody. TERRA FISH was performed the same way as in (A). DNA was stained by DAPI. The large DAPI-positive circle is the T. brucei nucleus, while the smaller DAPI-positive dot is the kinetoplast. All images are of the same scale, with the scale bar shown in the top right panel. (B) Northern blot of PF T. brucei total RNA. In both left and right panels, the EtBr-stained agarose gel images are shown on the left. Hybridization using the TbTR probe was done as a loading control (shown on the right). An 800 bp (TTAGGG)n-containing DNA fragment was labeled with radioactive dGTP in the absence of dCTP to generate a probe that was used to detect the (CCCUAA)n-containing TERRA (left). The same probe was labeled with radioactive dCTP in the absence of dGTP to generate a probe that was used to detect (UUAGGG)n-containing TERRA (right). Hybridizations were done at 50°C. 10 units of RNase A and 20 units of RNase One were added in the RNase treatment control samples.
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