20.500.12556/RUL-187790 Strukturni vpogled v interakcijo med 3'-UTR mRNA LRP5 in miR-629-3p Structural insight into the interaction between the 3'-UTR of LRP5 mRNA and miR-629-3p Koreceptor LRP5 sodeluje v kanonični Wnt/β-kateninski signalni poti, ki je pomembna za diferenciacijo osteoblastov in nastajanje kosti. Motnje v njegovem izražanju so povezane z boleznimi, kot so osteoporoza, hiperostoza in osteoporozni psevdogliomski sindrom. V magistrskem delu smo se osredotočili na uravnavanje izražanja LRP5 na ravni mRNA. V 3'-UTR mRNA LRP5 se nahaja z gvanini bogato zaporedje, ki se lahko zvije v G-kvadrupleks in vpliva na translacijo LRP5. V neposredni bližini tega zaporedja se hkrati veže več miRNA, ki so znani regulatorji translacije. Preučili smo, ali tvorba G-kvadrupleksa vpliva na dostopnost vezavnega mesta za miRNA ter kako vezava miRNA na mRNA vpliva na ravnotežje med G-kvadrupleksom in dupleksom. Z NMR in komplementarnimi spektroskopskimi metodami smo strukturno preučili z gvanini bogato zaporedje iz 3'-UTR mRNA LRP5 ter analizirali vpliv vezave izbranih miRNA. Najprej smo pokazali, da se z gvanini bogato zaporedje LRP5 neodvisno od dolžine preučevanega konstrukta v prisotnosti kalijevih ionov zvije v termično stabilen trikvartetni G-kvadrupleks s paralelno topologijo. Med izbranimi miRNA smo interakcijo z zaporedjem mRNA LRP5 zaznali predvsem pri miR-629-3p, katere vezavno mesto se prekriva z z gvanini bogatim območjem. S titracijami ter eksperimenti segrevanja in ohlajanja smo pokazali, da miR-629-3p vpliva na strukturno ravnotežje med G-kvadrupleksom in dupleksom ter ga postopoma pomakne proti dupleksu. Rezultati kažejo, da miR-629-3p najprej tvori bazne pare s 3'-podaljškom G-kvadrupleksa, nato pa se bazno parjenje postopoma razširi na preostali del z gvanini bogatega zaporedja, kar spremlja zmanjševanje deleža G-kvadrupleksa. Z načrtnim povečevanjem komplementarnosti med mRNA in modelnimi miRNA smo dodatno pokazali, da večje število komplementarnih baznih parov poveča delež dupleksa. Nazadnje smo z eksperimenti v lizatu celic HeLa pokazali, da se v pogojih makromolekulske gneče ohranita tako G-kvadrupleks kot dupleks. The LRP5 coreceptor participates in the canonical Wnt/β-catenin signalling pathway, which is important for osteoblast differentiation and bone formation. Dysregulation of its expression is associated with diseases such as osteoporosis, hyperostosis and Osteoporosis-Pseudoglioma Syndrome. Here, we focused on the regulation of LRP5 expression at the mRNA level. A guanine-rich (G-rich) sequence is located within the 3'-UTR of LRP5 mRNA and can fold into a G-quadruplex, affecting LRP5 translation. At the same time, several miRNAs, which are known regulators of translation, are predicted to bind in close proximity to this sequence. We investigated whether G-quadruplex formation affects the accessibility of the miRNA binding site and how miRNA binding to the mRNA is reflected in the G-quadruplex:duplex equilibrium. Using NMR and complementary spectroscopic methods, we structurally characterised the G-rich sequence from the 3'-UTR of LRP5 mRNA and analysed how its structure is affected by binding of selected miRNAs. First, we showed that, irrespective of the length of the construct, the G-rich LRP5 sequence in the presence of potassium ions folds into a thermally stable, three-layered G-quadruplex with a parallel topology. Among the selected miRNAs, miR-629-3p, whose binding site overlaps with the G-rich LRP5 sequence, showed the strongest interaction with the LRP5 mRNA sequence. Using titration as well as heating and cooling experiments, we demonstrated that miR-629-3p affects the structural equilibrium between the G-quadruplex and the duplex, gradually shifting it towards the duplex. The results indicate that miR-629-3p initially binds to the 3’-extension of the G-quadruplex and gradually unfolds it. By systematically increasing the complementarity between the mRNA and model miRNAs, we further demonstrated that a higher number of complementary base pairs increases the fraction of duplex in the sample. Finally, experiments in HeLa cell lysate showed that molecular crowding does not disrupt either the G-quadruplex or the duplex structure. G-kvadrupleks LRP5 miR-629-3p NMR spektroskopija G-quadruplex LRP5 miR-629-3p NMR spectroscopy true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-14 13:35:01 2026-09-14 13:35:08 2026-10-29 11:11:20 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25642 303052035 Moskric_Anja_-_Strukturni_vpogled_v_interakcijo_med_3_-UTR_mRNA_LRP5_in_miR-629-3p.pdf Moskric_Anja_-_Strukturni_vpogled_v_interakcijo_med_3_-UTR_mRNA_LRP5_in_miR-629-3p.pdf 1 4454384D6584C93FDDE4FF37F4C7B222 ead9f333b24455716c0a5e3dba1d8752362e4c4cfb7984650474baac87fbcd29 4ee42583-b030-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=247547 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0