Analiza tarč ORF1p, zaznanih z biotinsko identifikacijo, in optimizacija testa retrotranspozicije z vtRNA ter YRNA

Polanec, Klementina (author), Župunski, Vera (mentor)

Retrotranspozoni LINE1 so pri ljudeh edini še danes aktivni avtonomni mobilni genetski elementi. Predstavljajo kar 17 % celotnega genoma, sodelujejo pa tudi pri prenosu neavtonomnih retrotranspozonov. Element LINE1 zapisuje 3 proteine, in sicer ORF1p, ORF2p in ORF0p. ORF1p je RNA-vezavni protein z aktivnostjo šaperona za nukleinske kisline, ORF2p je endonukleaza in reverzna transkriptaza, funkcije ORF0p pa zaenkrat še ne razumemo dobro. Povišano raven retrotranspozicije LINE1 so opazili že pri več boleznih, vedno več raziskav pa povezuje LINE1 tudi z nekaterimi nevrodegenerativnimi boleznimi. Za razvoj terapevtskih učinkovin je ključno celostno razumevanje retrotranspozicije LINE1. Zato odkrivajo vedno več proteinov in RNA, ki interagirajo s komponentami LINE1, predvsem z ORF1p, in vplivajo na retrotranspozicijo. V prvem sklopu magistrskega dela smo s testom retrotranspozicije in meritvami na pretočnem citometru ugotovili, da RNY4 in vtRNA1-1 statistično značilno inhibirata retrotranspozicijo LINE1. Enak vpliv sta obe molekuli RNA imeli tako v celični liniji HeLa kot v celični liniji HEK293T, in sicer sta delež retrotranspozicije LINE1 znižali za približno eno četrtino. V drugem sklopu magistrskega dela smo analizirali rezultate masne spektrometrije po biotinski identifikaciji interakcijskih partnerjev ORF1p. Z imunodetekcijami po prenosih western smo v eluatih ORF1p-BioID2-HA po testih "pull-down" zaznali 5 potencialnih interakcijskih partnerjev ORF1p, in sicer IGF2BP1, TDP-43, ELAVL1, FUS in hnRNPK. Za te proteine smo s tem potrdili, da so interakcijski partnerji ORF1p. IGF2BP1 in ELAVL1 sta bila v celicah locirana na istih mestih kot prehodno izraženi ORF1p, v glavnem na obrobju mehurjastih tvorb ORF1p. Te bi lahko predstavljale začetne stopnje granulofagije ali endosome. Z analizo obogatenih pojmov genske ontologije smo za potencialne interakcijske partnerje ORF1p ugotovili, da se tako kot ORF1p nahajajo v stresnih granulah, citoplazmi, jedru in jedrcih. Večinoma gre za RNA-vezavne proteine, ki so udeleženi v različnih stopnjah metabolizma RNA. Rezultati prispevajo k boljšemu razumevanju delovanja LINE1 in dejavnikov, ki vplivajo na retrotranspozicijo.

• Language: Slovenian
• Keywords: retrotranspozon LINE1, ORF1p, vtRNA, YRNA, BioID2
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-146402
• COBISS.SI-ID: 158638595
• Publication date in RUL: 31.05.2023

Secondary language

• Language: English
• Title: Analysis of biotin identification targets of ORF1p and optimization of retrotransposition assay with vtRNA and YRNA

Abstract

Retrotransposon LINE1 is the only autonomous mobile genetic element still active in humans. It accounts for up to 17% of the human genome and helps mobilize nonautonomous retrotransposons. LINE1 element contains open reading frames for 3 proteins, namely ORF1p, ORF2p and ORF0p. ORF1p is an RNA-binding protein that acts as a nucleic acid chaperone, ORF2p is an endonuclease and a reverse transcriptase, whereas the function of ORF0p is not yet fully understood. Increased levels of LINE1 retrotransposition have been associated with several diseases, with a growing number of research linking LINE1 to certain neurodegenerative diseases. A comprehensive understanding of LINE1 retrotransposition is essential for the development of potential therapeutics. Therefore, it is important to discover new proteins and RNA molecules that interact with LINE1 components (especially ORF1p) and modulate retrotransposition. In the first part of this master's thesis we demonstrated by retrotransposition assay and flow cytometry that RNY4 and vtRNA1-1 have a statistically significant inhibitory effect on LINE1 retrotransposition. Both RNA molecules exhibited the same inhibitory effect in HeLa and HEK293T cell lines, decreasing the LINE1 retrotransposition by approximately 25%. In the second part of this master's thesis we analyzed the mass spectrometry results of biotin identification of ORF1p interaction partners. Using immunodetection after western blotting, we detected the presence of 5 potential ORF1p interaction partners (IGF2BP1, TDP-43, ELAVL1, FUS, hnRNPK) in ORF1p-BioID2- HA eluates from pull-down assays. Thus, these 5 proteins are true ORF1p interactors. IGF2BP1 and ELAVL1 colocalized with transiently expressed ORF1p, mostly at the edges of ring-shaped ORF1p formations. These formations could represent an initial phase of granulophagy or endosomes. Gene ontology term enrichment analysis revealed that potential ORF1p interaction partners are localized in stress granules, cytoplasm, nucleus, and nucleoli, just like ORF1p. Most of them are RNA-binding proteins involved in various steps of RNA metabolism. Our results contribute to a better understanding of LINE1 and the factors regulating retrotransposition.

• Keywords: retrotransposon LINE1, ORF1p, vtRNA, YRNA, BioID2