Guanine‐quadruplexes (G4s) are non‐canonical DNA structures that can regulate key biological processes such as transcription, replication and telomere maintenance in several organisms including eukaryotes, prokaryotes and viruses. Recent reports have identified the presence of G4s within the AT‐rich genome of Plasmodium falciparum, the protozoan parasite causing malaria. In Plasmodium, potential G4‐forming sequences (G4FS) are enriched in the telomeric and sub‐telomeric regions of the genome where they are associated with telomere maintenance and recombination events within virulence genes. However, there is a little understanding about the biological role of G4s and G4‐binding proteins. Here, we provide the first snapshot of G4‐interactome in P. falciparum using DNA pull‐down assay followed by LC–MS/MS. Interestingly, we identified ~24 potential G4‐binding proteins (G4‐BP) that bind to a stable G4FS (AP2_G4). Furthermore, we characterised the role of G‐strand binding protein 2 (PfGBP2), a putative telomere‐binding protein in P. falciparum. We validated the interaction of PfGBP2 with G4 in vitro as well as in vivo. PfGBP2 is expressed throughout the intra‐erythrocytic developmental cycle and is essential for the parasites in the presence of G4‐stabilising ligand, pyridostatin. Gene knockout studies showed the role of PfGBP2 in the expression of var genes. Taken together, this study suggests that PfGBP2 is a bona fide G4‐binding protein, which is likely to be involved in the regulation of G4‐related functions in these malarial parasites. In addition, this study sheds light on this understudied G4 biology in P. falciparum.

中文翻译：

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PfGBP2 是恶性疟原虫中的一种新型 G-四链体结合蛋白

鸟嘌呤四链体 (G4s) 是非规范 DNA 结构，可以调节包括真核生物、原核生物和病毒在内的多种生物体中的关键生物过程，如转录、复制和端粒维持。最近的报告已经确定了在恶性疟原虫（导致疟疾的原生动物寄生虫）的富含 AT 的基因组中存在 G4 。在疟原虫中，潜在的 G4 形成序列 (G4FS) 富集在基因组的端粒和亚端粒区域，它们与毒力基因内的端粒维持和重组事件相关。然而，对 G4s 和 G4 结合蛋白的生物学作用知之甚少。在这里，我们提供了恶性疟原虫G4-interactome 的第一个快照使用 DNA pull-down 分析，然后是 LC-MS/MS。有趣的是，我们鉴定了约 24 种潜在的 G4 结合蛋白（G4-BP），它们与稳定的 G4FS（AP2_G4）结合。此外，我们表征了 G 链结合蛋白 2 (PfGBP2) 的作用，它是恶性疟原虫中一种推定的端粒结合蛋白。我们在体外和体内验证了 PfGBP2 与 G4 的相互作用。PfGBP2 在整个红细胞内发育周期中表达，并且在 G4 稳定配体吡啶抑制素存在的情况下对寄生虫至关重要。基因敲除研究表明 PfGBP2 在var表达中的作用基因。综上所述，这项研究表明 PfGBP2 是一种真正的 G4 结合蛋白，它可能参与这些疟原虫 G4 相关功能的调节。此外，这项研究揭示了恶性疟原虫中这种未被充分研究的 G4 生物学。