Abstract
Trypanosoma brucei causes debilitating human African trypanosomiasis and evades the host’s immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. We previously showed that two interacting telomere proteins, TbTRF and TbTIF2, are essential for cell proliferation and suppress VSG switching by inhibiting DNA recombination events involving the whole active VSG expression site. We now find that TbTIF2 stabilizes TbTRF protein levels by inhibiting their degradation by the 26S proteasome, indicating that decreased TbTRF protein levels in TbTIF2-depleted cells contribute to more frequent VSG switching and eventual cell growth arrest. Surprisingly, although TbTIF2 depletion leads to more subtelomeric DNA double strand breaks (DSBs) that are both potent VSG switching inducers and detrimental to cell viability, TbTRF depletion does not increase the amount of DSBs inside subtelomeric VSG expression sites. Furthermore, expressing an ectopic allele of F2H-TbTRF in TbTIF2 RNAi cells allowed cells to maintain normal TbTRF protein levels for a longer frame of time. This resulted in a mildly better cell growth and partially suppressed the phenotype of increased VSG switching frequency but did not suppress the phenotype of more subtelomeric DSBs in TbTIF2-depleted cells. Therefore, TbTIF2 depletion has two parallel effects: decreased TbTRF protein levels and increased subtelomeric DSBs, both resulting in an acute increased VSG switching frequency and eventual cell growth arrest.
Highlights
Trypanosome brucei is a protozoan parasite that causes fatal African trypanosomiasis in humans and nagana in cattle
A transient TbTIF2 or TbTRF RNAi induction resulted in a significant increase in VSG switching frequency with most VSG switchers arising from subtelomeric gene rearrangements that resulted in the loss of the whole active expression sites (ESs) [18,19]
Northern blotting analysis showed that only the TbTIF2 mRNA was knocked-down by TbTIF2 RNAi, while TbTRF mRNA levels were not affected (Fig 1B), indicating that TbTIF2 is required for maintaining TbTRF protein levels
Summary
Trypanosome brucei is a protozoan parasite that causes fatal African trypanosomiasis in humans and nagana in cattle. TbTIF2 is an intrinsic component of the T. brucei telomere protein complex [19] It interacts tightly with the duplex telomere DNA binding factor, TbTRF, which has been shown to play an essential role in maintaining the terminal telomere structure [29]. We found that depletion of TbTIF2 resulted in a significant increase in the amount of DSBs at subtelomeres and subsequent elevated VSG switching frequency [19], demonstrating for the first time that telomere proteins play important roles in maintaining subtelomere integrity. Depletion of TbTRF did not increase the amount of DSBs inside the subtelomeric VSG ESs. expression of an ectopic TbTRF WT allele delayed TbTRF degradation, mildly improved cell growth, and partially suppressed the phenotype of elevated VSG switching frequency in TbTIF2 RNAi cells. Our observations indicate that increased amounts of subtelomeric DSBs and decreased TbTRF protein levels are two independent and parallel consequences of TbTIF2 depletion, both contributing to increased VSG switching frequencies and eventual cell growth arrest
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