Investigating non canonical DNA and RNA secondary structures: from their repair mechanism to their role in the miRNA processing pathway

APE1 is a protein mostly known for its role as a DNA repair enzyme, as it is involved in the BER pathway and it is the main endonuclease implicated in the repair of AP sites in DNA. AP sites, which affect both DNA and RNA, can be generated, in DNA, spontaneously or by the action of glycosylases, while their origin in RNA is still unknown. Recently, new roles of APE1 were discovered, mainly regarding DNA G4 biology and RNA metabolism. Indeed, some regions of DNA, as well as some RNA molecules, can form alternative secondary structures, influencing biological processes involving gene expression regulation, telomere maintenance and miRNA biogenesis. Within those structures, the most recognized are G4, in guanosine-rich strands, and iM, in the complementary cytosine-rich strands. Our laboratory and others have recently contributed to the characterization of APE1’s role towards DNA G4, demonstrating that APE1 can bind these structures and repair AP sites embedded within, but with lower efficiency compared to the canonical DNA duplex. Up to now, it is unknown whether APE1 may play any possible role in regulating other secondary structures, like G4 in RNA (rG4) or iM. Specifically, rG4 play roles in the miRNA maturation pathway, inhibiting the processing activity of the enzymes of the pathway. Starting from the considerations that a high number of miRNA precursors dysregulated upon APE1-depletion in cancer cells holds G4 forming sequences and that APE1 is already known to be involved in the miRNA processing pathway, we hypothesized a role of APE1 in the G4-dependent miRNA maturation pathway. As our working model, we chose the precursor form of miR-92b, which contains a well characterized rG4, which is present in a dynamic equilibrium with the canonical hairpin, influencing its biogenesis. Using different in vitro and cellular assays, we showed that APE1 can bind and modulate the folding of the rG4 contained in pre-mir-92b, with a mechanism strictly dependent on lysine residues present in its N-terminal region. APE1 cellular depletion impacted on the maturation process of miR-92b, mainly affecting its shuttling from nucleus to cytosol compartments. Lastly, the APE1-regulated rG4-containing miRNAs signature exhibited high prognostic significance in lung, cervical and liver tumors, suggesting potential targets for cancer therapy. As anticipated, another secondary structure folds in the G4 complementary cytosine-rich strand, called iM, which is also essential for telomere maintenance. iM processing biology is still poorly understood and it is presently unknown whether APE1 may interact and process AP sites embedded in these structures, thus investigations on these topics could be relevant for understanding the functional significance of genomic regions containing iM. In light of this, our work investigates APE1’s binding and processing capabilities toward native and damaged telomeric iM, specifically bearing AP sites in different positions of the iM structure. By employing orthogonal in vitro biochemical and biophysical techniques, we found that APE1 binds the telomeric iM-containing sequence. Endonuclease assays showed that APE1 cleavage efficiency depends on AP site position within iM, highlighting that those lesions, when present in the core are processed more efficiently than those in the loops. PLA analysis in HeLa and U2OS cells revealed the relevance of a novel interaction between APE1 with PCBP1, a well-known iM-folding modulator. PCBP1 binds iM with higher affinity than APE1 and inhibits its cleavage activity on damaged iM. Moreover, the depletion of APE1 or PCBP1 impairs their interaction with the shelterin components, affecting telomere length and highlighting the biological relevance of their crosstalk. All these results emphasize the role of APE1 on telomere stability, connecting its canonical DNA repair activity with the maintenance of non-canonical DNA secondary structures, through its interaction with PCBP1.