20.500.12556/RUL-173383 Interakcije med proteinom hnRNPH1 in RNA-ponovitvami GGGGCC, značilnih za ALS/FTD Interactions between hnRNP H1 protein and GGGGCC RNA repeats associated with ALS/FTD Amiotrofična lateralna skleroza (ALS) je smrtonosna nevrodegenerativna bolezen, katere vzrok je lahko ekspanzija heksanukleotidnih ponovitev G4C2 v genu C9orf72. Te ponovitve lahko tvorijo strukture G-kvadrupleksa (GQ). V magistrski nalogi smo želeli raziskati, ali protein hnRNPH1, ki veže RNA, interagira s temi ponovitvami in ali prednostno veže linearne ali GQ strukture. Klonirali in izrazili smo hnRNPH1 in protein PABPC1 v E. coli z uporabo His- in MBP-oznak za čiščenje. Proteine smo očistili z afinitetno in ionsko izmenjevalno kromatografijo, z masno spektrometrijo in prenosom western pa smo izvedli identifikacijo. Za oceno vezave smo uporabili metodo termoforeze na mikroskali (MST) in interferometrijo z biološkimi plastmi (BLI) z DNA- sondami, ki posnemajo ponovitve RNA. MST je pokazal, da se hnRNPH1 veže tako na linearne kot na GQ-oblike, z močnejšo afiniteto za razvito strukturo. Protein PABPC1 pa se z večjo afiniteto veže na nerazvite strukture. Metoda BLI pa potrebuje še dodatno optimizacijo. Naše ugotovitve kažejo, da se protein hnRNPH1 lahko prednostno veže na strukture GQ in jih potencialno razvija, kar lahko pripomore k razumevanju njegove vloge pri patologiji ALS, povezani s C9orf72. Prihodnje delo se bo osredotočilo na testiranje RNA-sond in dodatnih proteinskih konstruktov za nadaljnjo opredelitev te interakcije. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, often linked to a G4C2 hexanucleotide repeat expansion in the C9orf72 gene. These repeats can form G-quadruplex (GQ) structures, potentially contributing to disease pathology. In my master thesis project, we investigated whether the RNA-binding protein hnRNPH1 interacts with these repeats and whether it shows preference for linear or GQ structures. We cloned and expressed hnRNPH1 and control protein PABPC1 in E. coli, using His- and MBP-tags for purification. Proteins were purified via affinity and ion-exchange chromatography, with confirmation by mass spectrometry and Western blot. To assess binding, we used Microscale Thermophoresis (MST) and Bio-Layer Interferometry (BLI) with DNA probes mimicking RNA repeats. MST revealed that hnRNPH1 binds both linear and GQ forms, with stronger affinity for the linear structure. PABPC1 however binds with greater afinity to the linearized GQ form. BLI method requiers further optimisation. Our findings suggest that hnRNPH1 may preferentially bind and potentially unfold GQ structures, providing insight into its possible role in C9orf72- related ALS pathology. Future work will focus on testing RNA probes and additional protein constructs to further define this interaction. ALS C9ORF72 G-kvadrupleks hnRNPH1 PABPC1 kromatografija masna spektrometrija termoforeza interferometrija z biološkimi plastmi ALS C9ORF72 G-quadruplex hnRNPH1 PABPC1 chromatography mass spectrometry thermophoresis bio-layer interferometry true false false [G. Turk] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-17 07:17:23 2025-09-17 07:17:27 2025-11-08 03:51:55 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 616-004:544.17:577.112(043.2) 264658 249242627 51376.pdf 51376.pdf 1 B4F771AB44356817E05BBF58E542881A 061fe164c1a9288e67718066656d77ae25ecb9c4b437fd1c191dcca1b7af6926 6dc13ebd-9385-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=217793 Biotehniška fakulteta 0 0 0