Title In vitro system for the detection of ADAR editing of HSV-1 miRNAs Title (croatian) In vitro sustav za detekciju ADAR uređivanja miRNA kod HSV-1 Author Sara Patačko Mentor Igor Jurak (mentor) Committee member Ivana Munitić (član povjerenstva) Committee member Katarina Kapuralin (predsjednik povjerenstva) Granter University of Rijeka Defense date and country 2024-09-23, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Post-transcriptional changes in the RNA nucleotide sequences, known as RNA editing, include insertion, deletion, and substitution of nucleotides. Editing can be present in both protein-coding and non-coding transcripts, impacting their stability, splicing, ability to regulate gene expression, and it can even lead to the recoding of proteins. Conversion of adenosine to inosine (A→I) in dsRNAs, catalyzed by the adenosine deaminase acting on RNA (ADAR) protein family, is the most prevalent form of RNA editing. Herpes simplex virus 1 (HSV-1), a highly prevalent human pathogen, encodes for over 20 miRNAs; and during latency, the only present transcripts are latency-associated transcripts (LATs) and miRNAs. Our group previously detected A→I editing events in HSV-1 miRNAs from latently infected human terminal ganglia. Highly expressed miR-H2, whose target is ICP0, an immediate early viral gene crucial for viral replication, was most frequently edited. However; the molecular mechanism of editing still needs to be unraveled. Thus, we generated an in vitro system for detecting ADAR editing events in HSV-1 miRNAs, based on the co-transfection of miRNA-expressing constructs with different ADAR-expressing plasmids. The backbone for miRNA expression plasmids (ps-Cassette) was successfully produced by cloning synthetic expression cassette into pEGFP-N1. Furthermore, the miRNA expression plasmid pS-miR-H2 and the editing positive control plasmid pS-miR-376a were generated by cloning miRNA coding fragments into the pS-cassette. Expression of the pS-miR plasmids transfected into HEK293T cells was not confirmed because of plasmid DNA contaminations. Additionally, Western blot analysis revealed high expression of endogenous ADAR1 p110 in HEK293 cells indicating potential background contribution to editing, which is why ADAR1 KO cells need to be used. Our system still has to be validated and there is room for improvement in the future. Nevertheless, the successful production of HSV-1 miRNA-expressing plasmids represents a significant advancement in detecting the molecular mechanism behind ADAR editing of HSV-1 miRNAs.
Abstract (croatian) Post-transkripcijske modifikacije u slijedu RNA nukleotida, poznate kao uređivanje RNA, uključuju inserciju, deleciju i substituciju nukleotida. Uređivanje može biti prisutno u protein-kodirajućim i nekodirajućim transkriptima, te posljedično utječe na njihovu stabilnost, prekrajanje, sposobnost regulacije genske ekspresije te čak može dovesti do rekodiranja proteina. Konverzija adenozina u inozin (A→I) u dvolančanim RNA, katalizirana adenozin deaminazama koje djeluju na RNA (ADAR), najučestalija je forma uređivanja RNA. Herpes simplex virus tip 1 (HSV-1), visoko prevalentni humani patogen, kodira više od 20 mikroRNA (miRNA). Zanimljivo je da su tijekom latentne faze jedino prisutni transkripti povezani s latencijom (LAT) te miRNA. Naša istraživačka skupina je nedavno detektirala A→I uređivanja u HSV-1 miRNA u latentno inficiranim humani terminalnim ganglijima. Visoko eksprimirana miR-H2, čija je meta ICP0, neposredno rani viralni gen ključan za viralnu replikaciju, je bila najčešće uređivana. Međutim, molekularni mehanizam ovog uređivanja još uvijek nije otkriven. Obzirom na to, generirali smo in vitro sustav za detekciju ADAR uređivanja u miRNA HSV-1, baziran na kotransfekciji miRNA-eksprimirajućih kostrukata s različitim ADAR-eksprimirajućim plazmidima. Okosnica miRNA ekspresijskog plazmida (ps-Cassette) je uspješno producirana kloniranjem sintetske ekspresijske kazete u pEGFP-N1 plazmid. Nadalje, miRNA ekspresijski plasmidi pS-miR-H2 te pS-miR-376a, koji je ujedno i pozitivna kontrola editiranja, su generirani kloniranjem miRNA kodirajućih fragmenata u pS-Cassette. Ekspresija pS-miR plazmida transfeciranih u HEK293T stanice nije potvrđena zbog kontaminacije izolirane RNA s plazmidnom DNA. Western blot analiza pokazala je kako je ekspresija endogenog ADAR1 p110 u HEK293 stanicama iznimno visoka što ukazuje da bi mogao neposredno doprinijeti RNA uređivanju u našem sustavu. Stoga je u budućnosti potrebno koristiti stanice s utišanim genom za ADAR1. Ovaj in vitro sustav još treba biti validiran te postoji mjesta za napredak u budućnosti. Usprkos tome, uspješna produkcija HSV-1 miRNA-ekspresijskih plazmida predstavlja značajan napredak u detekciji molekularnog mehanizma koji se krije iza ADAR uređivanja HSV-1 miRNA.
Keywords
herpes simplex virus 1 (HSV-1)
micro-RNA
RNA editing
ADAR
Keywords (english)
herpes simplex virus 1 (HSV-1)
mikro-RNA
uređivanje RNA
ADAR
Language english URN:NBN urn:nbn:hr:193:798000 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Embargoed access Terms of use Created on 2024-09-23 23:16:48
