Identification of G-quadruplex-interacting proteins within the hexameric repeat of the XDP-associated SVA retrotransposon

X-linked Dystonia-Parkinsonism (XDP) is an adult-onset neurodegenerative disease endemic to Panay, Philippines. The clinical course of XDP is marked by progressive motor impairment, resulting from neurodegeneration, and death as outcome. At present, there is no effective cure, underscoring the urgent need for new treatment strategies. All reported XDP patients inherit a shared haplotype in the Xq13.1 region of the X chromosome which includes a disease-specific antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in the TATA-binding protein associated factor 1 (TAF1) gene, which encodes a core subunit of the transcription factor II D (TFIID) complex. SVAs are composite retrotransposons specific to hominids, comprising (5’-3’) hexanucleotide (CCCTCT)n repeats (HEX), two antisense Alu-like fragments, a variable number of GC-rich tandem repeats (VNTR), a short interspersed nuclear element region (SINE) derived from the 3’ LTR of the HERV-K10 retroviral element, and a poly(A) tail. Notably, the HEX is highly amplified in the XDP SVA, ranging from approximately 30 to 50 repeats among patients, whereas canonical SVAs carry only 3 to 10 repeats. In XDP, longer HEX tracts correlate with earlier disease onset and more severe symptoms, suggesting a role of HEX amplification in XDP pathogenesis. Given the high GC content, especially in the HEX domain, the XDP SVA is prone to fold into G-quadruplexes (G4s), non-canonical nucleic acid secondary structures that play critical roles in essential biological processes. By contrast, the SINE domain within the same SVA does not form G4s and was therefore used as a negative control in this work. Previous studies have shown that the XDP SVA G4s could transiently stall RNA polymerase II, thereby reducing the production of full-length TAF1 mRNA, a characteristic feature of XDP patients. In this context, the aim of this thesis was to identify proteins that interact with G4s within the HEX domain to improve our understanding of XDP molecular mechanisms. To this end, protein pull-down assays were performed using nuclear protein extracts from Neural Progenitor Cells (NPCs) derived from XDP patients. Two types of biotinylated DNA baits were employed: btn-ss-Hex4, a single-stranded oligonucleotide representing the minimal G4-forming motif (AGAGGG)4; and btn-ds-HEX41, a double-stranded sequence representing the full-length HEX domain (GGGAGA)41. In parallel, two biotinylated DNA sequences were included as negative controls to exclude proteins that bind DNA independently of G4 formation: btn-ss-Rnd, a randomized version of btn-ss-Hex4; and btn-ds-SINE, a double-stranded sequence representing a SINE domain unrelated to HEX of the XDP SVA. We proved with biophysical studies that neither of the negative control baits adopt a G4 in our working conditions. Bait-bound proteins were isolated and analyzed by mass spectrometry to identify putative interactors of HEX G4s. Notably, among the proteins enriched for the G4-forming btn-ss-Hex4 bait, we detected the well-known G4-resolving helicase DHX36. This finding was further validated through pull-down western blot analysis.