Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability. At their level, particular forms of chromatin may be found. G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4’s biological significance. Very recently, scientists developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Implementing this innovative strategy in live cells, they unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. After narrowing down the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, GAR1, ILF3) in vitro, it was found that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3′ splice site.
The corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. Another telomeric structures is the so called R-loop. Dysregulated R-loops can cause stalled replication forks and telomere instability. The telomeric repeat-containing RNA (TERRA) that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop. In tumor cells, R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres (ALT) pathway. However, how R-loops are recognized and regulated, is still not well understood, particularly at telomeres. In another investigation, researchers from Sun Yat-sen University, Guangzhou, used proximity-dependent biotin identification (BioID) technology to identify the ILF3 interactome and discovered that ILF3 interacts with several DNA/RNA helicases.
Among those, there is DHX9; this interaction suggests that ILF3 may facilitate the resolution of telomeric R-loops, thereby preventing abnormal homologous recombination and maintaining telomere homeostasis. The research team discovered that ILF3 exhibits selective interact with telomeric R-loops, thereby safeguarding telomeres against aberrant homologous recombination. ILF3 loss of function consequently elevates TERRA levels, triggering the accumulation of R-loops at telomeres. This accumulation induces DNA damage response (DDR) and telomere dysfunction, characterized by telomere fragility and the presence of extra-chromosomal telomere fragments, which may in turn activate the ALT pathway. ILF3 potentially acts as a reader for telomeric R-loops, aiding in the prevention of aberrant homologous recombination and the maintenance of telomere homeostasis.
These results support that ILF3 interacts with telomeric RNA:DNA hybrid structures such as R-loops and promotes the resolution or inhibits excessive accumulation of R-loops through the RNA helicase DHX9. This research provides new insights into the regulation of telomeric R-loops and the mechanisms that maintain telomere homeostasis, with implications for aging biology. Telomeres do not only have DNA or RNA components; there is telosome (also called shelterin) which is basically formed by the six constitutive proteins TRF1, TRF2, TIN2, RAP1, TPP1 and POT1. Moreover elomeres can be elongated through the telomerase holoenzyme that consists of the catalytic subunit TERT, the RNA template TERC and accessory proteins. The telomeric RNA TERRA is transcribed by RNA polymerase II from the C-rich strand of the telomeric subregion. Overexpression of TERRA inhibits telomerase activity and induces cell senescence.
TERRA and telomeric R-loop levels are upregulated in and human ALT cancer cell lines, leading to the high frequency of homologous recombinaton. In ALT cells, R-loops are found to inhibit ROS-induced telomeric DNA breaks through the CSB-RAD52-POLD3 pathway. This grants the repair of these genomic regions and avoiding responses leading to oncogenic destabilization. TERRA recruitment of PRC2 is responsible for catalyzing histone H3 trimethylation on residue K27 to ensure heterochromatin assembly on telomere. Beside, ILF3 loss leads to excessive R-loop formation, DNA damage response activation, and increased extra-chromosomal telomere fragments (C-circles) and aberrant homologous recombination. In senescent human cells, the expression of inflammatory cytokines is upregulated, which is known as the senescence-associated secretory phenotype (SASP). ILF3 helps maintain the youthful state of cells by inhibiting the translation of these SASP proteins.
Consistently, ILF3 is also down-regulated during replicative senescence, suggesting possible roles of ILF3 in tumorigenesis. Altogether, these informations demonstrate the important role of G4 quadruplexes, the TERRA telomeric RNA and the R-loops in telomere homeostasis. A deeper understanding of the kinetics of telosome with its own folds will give scientists more hints about crucial biological processes, like aging and cancer.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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