20.500.12556/RUL-184111 In vitro analiza fazne ločitve parapegnih proteinov in vpliva heksanukleotidnih RNA ponovitev mutiranega C9orf72 In vitro analysis of phase separation of paraspeckle proteins and the impact of hexanucleotide RNA repeats of mutated C9orf72 Parapege so nemembranski organeli, katerih ogrodje predstavlja dolga nekodirajoča RNA NEAT1 (jedrni prepis 1 združka parapeg). Na to ogrodje se vežejo specifični RNA-vezavni proteini, kot so protein spojen v sarkomu (FUS), protein brez domene POU, ki se veže na oktamer (NONO) in faktor bogat s prolini (SFPQ). Ti proteini preko procesa fazne ločitve tekoče-tekoče (LLPS) z molekulami NEAT1 tvorijo sferične kondenzate. V jedrne parapege se ob stresu lahko rekrutira tudi poli(A)-vezavni protein citoplazme 1 (PABPC1), ki sicer deluje kot gradnik stresnih granul v citoplazmi. Pri amiotrofični lateralni sklerozi (ALS) pride do mutacije v genu C9orf72, ki se kaže kot povečano število heksanukleotidnih ponovitev (G$_4$C$_2$)$_n$. Nastale mutirane molekule RNA v jedru tvorijo patološke agregate, ki delujejo kot alternativno ogrodje za proteine parapeg. To vodi v sekvestacijo, kar zmanjša njihovo razpoložljivost za celične procese. V diplomskem delu smo preučevali interakcije med proteini parapeg (FUS, PABPC1, NONO in SFPQ) ter RNA konstruktom (G$_4$C$_2$)$_{48}$ pri različnih molskih razmerjih, s čimer smo želeli dobiti vpogled v naravo njihovih interakcij. Z meritvami turbidnosti in analizo NaDS-PAGE smo potrdili vpliv RNA konstrukta (G$_4$C$_2$)$_{48}$ na topnost proteinov, kar nakazuje na interakcije med proteini in RNA. Molsko razmerje je bilo ključno pri proteinu FUS, kjer je višja koncentracija RNA bistveno zmanjšala agregacijo. Za nadaljnje raziskave je potrebno analize interakcij izvesti še s preostalimi RNA-vezavnimi proteini in pri drugačnih pogojih. Paraspeckles are membraneless organelles scaffolded by the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). Specific RNA-binding proteins, such as fused in sarcoma (FUS), non-POU domain-containing octamer-binding protein (NONO) and splicing factor proline and glutamine rich (SFPQ), bind to this RNA scaffold. Through the process of liquid-liquid phase separation (LLPS), these proteins interact with NEAT1 molecules to form spherical condensates. Upon stress, poly(A)-binding protein cytoplasmic 1 (PABPC1), which normally acts as a building block of cytoplasmic stress granules, can also be recruited to nuclear paraspeckles. In amyotrophic lateral sclerosis (ALS), mutations in the C9orf72 gene lead to an expanded number of hexanucleotide repeats (G$_4$C$_2$)$_n$. The resulting mutant RNA molecules form pathological aggregates in the nucleus, which act as an alternative scaffold for paraspeckle proteins. This leads to their sequestration thereby reducing their availability for essential cellular processes. In this bachelor's thesis, we investigated the interactions between paraspeckle proteins (FUS, PABPC1, NONO, and SFPQ) and the RNA construct (G$_4$C$_2$)$_{48}$ at various molar ratios to gain insight into the nature of their interactions. By measuring turbidity and performing NaDS-PAGE analysis, we confirmed the effect of the (G$_4$C$_2$)$_{48}$ RNA construct on protein solubility, indicating RNA-protein interactions. The molar ratio proved to be crucial for the FUS protein, where a higher RNA concentration significantly reduced aggregation. For future research, interaction analyses should be performed with the remaining RNA-binding proteins under different experimental conditions. parapege RNA-vezavni proteini ALS RNA konstrukt (G$_4$C$_2$)$_{48}$ paraspeckle RNA-binding proteins ALS (G$_4$C$_2$)$_{48}$ RNA construct true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-06-29 08:55:03 2026-06-29 08:55:19 2026-07-30 13:12:53 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 26098 286471939 Skarabot_Anja_-_In_vitro_analiza_fazne_locitve_parapegnih_proteinov_in_vpliva_heksanukleotidnih_.pdf Skarabot_Anja_-_In_vitro_analiza_fazne_locitve_parapegnih_proteinov_in_vpliva_heksanukleotidnih_.pdf 1 1AF74E9ECE01EA0AB2F3C74FA2DA5675 d50d3bbe0f8343dc5c18dc8c0da4422431f15e415ae433086b7b68059473155d 650f666e-7387-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236586 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0