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Vpliv RNA z razširjenimi ponovitvami na proces ločevanja faz
ID Ruter, Petra (Author), ID Plavec, Janez (Mentor) More about this mentor... This link opens in a new window, ID Lah, Jurij (Comentor)

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Abstract
Biomolekularni kondenzati so vrsta brezmembranskih organelov, ki nastanejo preko ločitve tekočih faz in vsebujejo pretežno proteine in nukleinske kisline. V kondenzatih se vzpostavi kemijsko različno okolje zaradi selektivne vključitve nekaterih in izključitve drugih makromolekul. Kondenzati v celici lahko opravljajo večje število vlog: ločitev celične vsebine s transportom in lokalizacijo nukleinskih kislin, vzpostavitev ugodnega okolja za izvedbo katalitičnih procesov, shranjevanje izbranih molekul in odziv na stres. Mutacije ključnih komponent ali deregulacija nastanka kondenzatov vodijo v patološka stanja, kot so nevrodegenerativne bolezni in miopatije, na primer ALS, Huntingtonova in Alzheimerjeva bolezen ter frontotemporalna demenca. Pri razvoju teh bolezni je bolje raziskana vloga proteinov, manj pa je znanega o prispevku RNA, ki naj bi predstavljala ogrodje za vezavo proteinov, interakcije RNA–RNA pa neposredno vplivale na sprožitev ločitve tekočih faz. Zaradi tega je potrebno pridobiti več podrobnih podatkov o lastnostih RNA, potrebnih za nastanek različnih biomolekularnih kondenzatov, ter razumeti odvisnost sistema od vrste kemijskih faktorjev. V okviru magistrskega dela smo izbrali osem različnih zaporedij RNA z razširjenimi ponovitvami (G$_4$C$_2$, CAG, CUG, CCUG, CGG, AUUCU, UGGAA in GGCCUG), ki se pojavljajo pri nevrodegenerativnih boleznih in miopatijah, in opazovali vpliv lastnosti RNA (zaporedje, dolžina, nastanek struktur višjega reda) in okolja (koncentracija kalijevih in magnezijevih kationov, koncentracija RNA, pH, dodatek polietilenglikola) na proces ločitve tekočih faz s pomočjo UV spektroskopije. Z meritvami turbidnosti smo opazili, da dolžina ponovitev v zaporedju ne vpliva na proces ločitve faz. Opazimo pa vpliv števila ponovitev na povečano turbidnost. Prisotnost kalijevih kationov povzroči povečano turbidnost pri zaporedjih (UGGAA)$_n$ in (CGG)$_n$, kjer kalijevi kationi tudi stabilizirajo predvidene strukture višjega reda. Magnezijevi kationi povzročijo povečano turbidnost predvsem pri zaporedjih (G$_4$C$_2$)$_n$ in (GGCCUG)$_n$. Višja koncentracija RNA povzroči povečano turbidnost pri (UGGAA)$_n$, (G$_4$C$_2$)$_n$, (CGG)$_n$, (GGCCUG)$_n$ in (CAG)$_n$, dodatek polietilenglikola pa povzroči povečano turbidnost pri (G$_4$C$_2$)$_n$, (CGG)$_n$ in (GGCCUG)$_n$.

Language:Slovenian
Keywords:RNA z razširjenimi ponovitvami, biomolekularni kondenzati, proces ločitve tekočih faz, turbidnost
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2024
PID:20.500.12556/RUL-159170 This link opens in a new window
COBISS.SI-ID:201504771 This link opens in a new window
Publication date in RUL:02.07.2024
Views:48
Downloads:5
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Secondary language

Language:English
Title:Induction of phase separation phenomena by RNA expansion repeats
Abstract:
Biomolecular condensates are a type of membrane-less organelles formed by liquid-liquid phase separation, containing mainly proteins and nucleic acids. In condensates, a chemically different environment is created by the selective inclusion of some macromolecules and the exclusion of others. Condensates can perform a number of roles in the cell: separating cellular contents by transporting and localising nucleic acids, creating a favourable environment for catalytic processes, storing selected molecules, and responding to stress. Mutations in key components or deregulation of condensate formation lead to pathological conditions such as neurodegenerative diseases and myopathies, for example ALS, Huntington's disease, Alzheimer's disease and frontotemporal dementia. The role of proteins in the development of these diseases has been intensively explored, but less is known about the contribution of RNA, even though RNA is thought to provide a scaffold for protein binding, and RNA–RNA interactions directly trigger the initiation of liquid-liquid phase separation. This calls for a more detailed exploration of the RNA properties required for the formation of the different biomolecular condensates and an understanding of the dependence of the system on a range of chemical factors. As a part of the MSc thesis, we selected eight different RNA sequences with expansion repeats (G$_4$C$_2$, CAG, CUG, CCUG, CGG, AUUCU, UGGAA and GGCCUG) occurring in neurodegenerative diseases and myopathies to observe the influence of the RNA properties (sequence, length, formation of higher order structures) and the environment (concentration of potassium and magnesium ions, concentration of the RNA, pH, addition of polyethylene glycol) on the process of separation of the liquid phases by UV spectroscopy. By measuring turbidity we observed that the length of repeats in the sequence does not affect the phase separation process. However, the effect of the number of iterations on the increased turbulence is noticeable. The presence of potassium ions causes increased turbidities for the (UGGAA)$_n$ and (CGG)$_n$ sequences, where potassium ions also stabilise the predicted higher-order structures. Magnesium ions cause increased turbidity, especially for the (G$_4$C$_2$)$_n$ and (GGCCUG)$_n$ sequences. High RNA concentration has a positive effect on turbidity for (UGGAA)$_n$, (G$_4$C$_2$)$_n$, (CGG)$_n$, (GGCCUG)$_n$ and (CAG)$_n$, while the addition of polyethylene glycol results in an increased turbidity for (G$_4$C$_2$)$_n$, (CGG)$_n$ and (GGCCUG)$_n$.

Keywords:: RNA expansion repeats, biomolecular condensates, liquid-liquid phase separation, turbidity

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