Vzpostavitev in vitro sistema za preučevanje vloge RNA pri procesu ločitve tekočih faz z RNA-oligonukleotidi Efl, Sulf in Wdr

Kogoj, Lena (author), Plavec, Janez (mentor)

Nemembranski organeli ali biomolekulski kondenzati, na primer jedrce in stresne granule, nastanejo s procesom ločitve tekočih faz (LLPS). V celicah opravljajo zelo pomembne funkcije, nepravilnosti v zvezi z njihovim delovanjem pa vodijo v bolezni, kot so nevrodegenerativne bolezni, določene vrste raka, virusne okužbe in dilativna kardiomiopatija, zato se znanost njihovemu raziskovanju v zadnjem času intenzivno posveča. V svoji diplomski nalogi se osredotočam na biomolekulske kondenzate, ki so ključni pri diferenciaciji naivnih mišjih embrionalnih matičnih celic, njihovo središče pa predstavljajo dolge nekodirajoče RNA Efl1, Sulf2 in Wdr62. Namen diplomskega dela je vzpostaviti in vitro rekonstituiran sistem, ki bi omogočil strukturne študije LLPS z NMR-spektroskopijo. Z bioinformatsko analizo smo na podlagi napovedi sekundarne strukture izbrali kratka oligonukleotidna zaporedja Efl, Sulf in Wdr, izvirajoča iz dolgih nekodirajočih RNA Efl1, Sulf2 in Wdr62. Poliakrilamidna gelska elektroforeza (PAGE) pod nativnimi pogoji in NMR-spektroskopija sta nam pokazali, da so oligonukleotidi v kislem in nevtralnem pH v nerazviti obliki, kar pomeni, da LLPS ne more poteči na osnovi interakcij med strukturnimi elementi RNA. S testi turbidnosti in svetlobno mikroskopijo smo določili, da kombinacije RNA oligonukleotidov, peptidov in različnih soli v večini primerov vodijo v agregacijo. Kljub temu nam je uspelo najti pogoje za potek LLPS, in sicer pri pogojih 20-75 µM RNA, 150-200 µM RNA-prepoznavnega motiva 3 proteina PTBP1, 3-5 mM Mg$^{2+}$ in 10-% masnega deleža PEG. Vpliva pH in Na$^+$ oziroma K$^+$ ionov na potek LLPS nismo opazili. Identiteto kapljic smo potrdili s testom raztapljanja s povečevanjem ionske moči. V povsem novem makromolekulskem sistemu smo tako našli pogoje, pri katerih poteče LLPS, pri čemer smo identificirali vplive različnih dejavnikov na stanje v sistemu. Stabilnost in ponovljivost vzpostavitve preučevanega sistema nam bosta omogočili strukturno analizo z NMR-spektroskopijo, ki bo dala pomemben uvid v nastanek in delovanje biomolekulskih kondenzatov, vključenih v embrionalni razvoj.

• Language: Slovenian
• Keywords: biomolekulski kondenzati, proces ločitve tekočih faz, svetlobna mikroskopija, UV-Vis spektroskopija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-170202
• COBISS.SI-ID: 242129923
• Publication date in RUL: 02.07.2025

Secondary language

• Language: English
• Title: Establishment of an in vitro system for studying the role of RNA in the process of liquid-liquid phase separation with RNA oligonucleotides Efl, Sulf and Wdr

Abstract

Membraneless organelles, also known as biomolecular condensates, such as the nucleolus and stress granules, are formed through a process called liquid–liquid phase separation (LLPS). These structures perform essential functions in cells, and malfunctions in their formation or behavior are linked to diseases including neurodegenerative disorders, certain types of cancer, viral infections, and dilated cardiomyopathy. Consequently, biomolecular condensates have recently become a major focus of scientific research. In my thesis, I focus on biomolecular condensates critical for the differentiation of naive mouse embryonic stem cells, which are centered around long non-coding RNAs named Efl, Sulf, and Wdr. The aim of this work is to establish an in vitro reconstituted system that enables structural studies of LLPS using NMR spectroscopy. Based on predicted secondary structures we selected short oligonucleotide sequences derived from Efl, Sulf, and Wdr. Native PAGE and NMR spectroscopy revealed that these oligonucleotides remain unstructured at acidic and neutral pH, indicating that LLPS cannot occur through RNA structural interactions alone. Using turbidity assays and light microscopy, we found that most combinations of RNA oligonucleotides, peptides, and various salts lead to aggregation. Nevertheless, we successfully identified conditions that support LLPS, specifically, 20–75 µM RNA, 150–200 µM RNA recognition motif 3 (RRM3) of the PTBP1 protein, 3–5 mM Mg²⁺, and 10 % (w/w) PEG. We observed no significant effect of pH or Na⁺/K⁺ ions on LLPS. The identity of the resulting droplets was confirmed using a dissolution by increasing ionic-strength assay. In this novel macromolecular system, we established conditions under which LLPS occurs and identified the influence of various factors on the system’s behavior. The stability and reproducibility of this in vitro system will enable structural analysis via NMR spectroscopy, offering valuable insight into the formation and function of biomolecular condensates involved in embryonic development.

• Keywords: biomolecular condensates, light microscopy, liquid-liquid phase separation, UV-Vis spectroscopy