20.500.12556/RUL-184483 Vpliv RNA-ponovitev (G$_4$C$_2$)$_{48}$ na ločitev faz tekoče-tekoče v raztopinah proteinov DAZAP1 in HNRNPH3 The effect of (G$_4$C$_2$)$_{48}$ RNA repeats on liquid–liquid phase separation of DAZAP1 and HNRNPH3 Heksanukleotidne RNA-ponovitve GGGGCC (G$_4$C$_2$) so vpletene v razvoj amiotrofične lateralne skleroze in frontotemporalne demence. Nanje se vežejo različni RNA-vezavni proteini, s čimer pride do njihove sekvestracije ali celo agregacije. Ker so ti proteini pogosto transkripcijski faktorji, to vpliva na uravnavanje izražanja genov. Po strukturi in funkciji so ti skupki RNA in proteinov podobni parapegam, jedrnim nemembranskim organelom, ki nastanejo kot odziv na celični stres in uravnavajo izražanje genov s sekvestracijo proteinov. Dva izmed proteinov, ki se nahajata v parapegah, sta z DAZ povezan protein 1 (DAZAP1) in heterogeni jedrni ribonukleoprotein H3 (HNRNPH3). RNA prepoznavni motivi in strukturno neurejene regije, ki jih vsebujeta, omogočajo ločitev faz tekoče tekoče, s katero nastanejo parapege. Preverili smo, ali RNA-ponovitve (G$_4$C$_2$)$_{48}$ povzročijo ločitev faz tekoče-tekoče teh dveh proteinov. Za izražanje zapisov za DAZAP1 in HNRNPH3 v E. coli smo pripravili rekombinantna konstrukta na osnovi vektorja pJ4M, ki je vseboval zapisa za C-končno fuzijo maltozo vezavnega proteina in heksahistidinsko oznako. Po indukciji izražanja smo proteina izolirali z nikljevo afinitetno kromatografijo. RNA-ponovitve (G$_4$C$_2$)$_{48}$ smo pripravili s transkripcijo in vitro. Ločitev faz tekoče-tekoče smo opazovali z mikroskopijo z diferencialnim interferenčnim kontrastom (DIC). Ugotovili smo, da RNA ponovitve (G$_4$C$_2$)$_{48}$ povzročijo ločitev faz tekoče-tekoče tako pri DAZAP1 kot tudi pri HNRNPH3. Poleg tega se DAZAP1 fazno loči tudi v prisotnosti 5-% polietilen glikola, ki posnema učinek molekulskega nagnetenja, značilnega za celico. Za ločitev faz tekoče-tekoče pri HNRNPH3 je bila potrebna bistveno višja koncentracija RNA kot pri DAZAP1, fazno ločene kapljice pa so bile po velikosti majhne in prisotne v nizkem številu, zato bi rezultate morali preveriti še z drugimi metodami. Eksperiment bi bilo smiselno ponoviti tudi z uporabo fluorescenčno označenih proteinov, s čimer bi lahko potrdili vezavo DAZAP1 in HNRNPH3 na RNA ponovitve (G$_4$C$_2$)$_{48}$ ter preučili njuno morebitno kolokalizacijo v celici. Hexanucleotide RNA repeats GGGGCC (G$_4$C$_2$) are implicated in the development of amyotrophic lateral sclerosis and frontotemporal dementia. They can sequester or promote the aggregation of various RNA-binding proteins, many of which act as transcription factors, thereby disrupting gene regulation. The structure and function of these RNA protein granules resemble that of paraspeckles – membraneless nuclear organelles that form in response to cellular stress and also regulate gene expression via protein sequestration. Two key paraspeckle proteins are DAZ associated protein 1 (DAZAP1) and heterogeneous nuclear ribonucleoprotein H3 (HNRNPH3). Both contain RNA recognition motifs and structurally disordered regions that facilitate liquid-liquid phase separation, the mechanism underlying paraspeckle formation. To investigate whether (G$_4$C$_2$)$_{48}$ RNA repeats cause liquid-liquid phase separation of DAZAP1 and HNRNPH3, we expressed DAZAP1 and HNRNPH3 with C-terminal fusion of maltose binding protein and His-tag using vectors we prepared by cloning the nucleotide sequences of these proteins into pJ4M. Expressed proteins were purified using nickel affinity chromatography. (G$_4$C$_2$)$_{48}$ RNA repeats were prepared by in vitro transcription. Liquid liquid phase separation was observed with differential interference contrast (DIC) microscopy. We found that (G$_4$C$_2$)$_{48}$ RNA repeats promote liquid-liquid phase separation of both DAZAP1 and HNRNPH3. Additionally, DAZAP1 underwent phase separation in the presence of 5% polyethylene glycol which mimics the effects of molecular crowding found in the cell. In contrast to DAZAP1, HNRNPH3 required substantially higher concentrations of RNA to undergo phase separation, while also producing fewer phase separated droplets that were smaller in size. These observations suggest that further validation using complementary methods is required. Future experiments employing fluorescently tagged proteins could help confirm binding of DAZAP1 and HNRNPH3 to (G$_4$C$_2$)$_{48}$ RNA repeats and assess their potential colocalization. DAZAP1 HNRNPH3 ločitev faz tekoče-tekoče parapege RNA-ponovitve DAZAP1 HNRNPH3 liquid-liquid phase separation paraspeckles RNA repeats true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-07-08 10:45:01 2026-07-08 10:45:15 2026-08-20 12:34:21 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25983 288327683 Susnik_Rok_-_Vpliv_RNA-ponovitev_(G4C2)48_na_locitev_faz_tekoce-tekoce_v_raztopinah_proteinov_DA.pdf Susnik_Rok_-_Vpliv_RNA-ponovitev_(G4C2)48_na_locitev_faz_tekoce-tekoce_v_raztopinah_proteinov_DA.pdf 1 91200546C7970B657365A255F4C159B8 7050154b8f832899e6b14de8680c628ea7d271a0bf589acc0194ca60632fde45 39712aa6-7aa9-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237317 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0