Vpliv proteina TDP-43 na retrotranspozicijo LINE-1 v sesalskih celičnih kulturah

Korošec, Tinkara (author), Župunski, Vera (mentor)

Mobilni elementi DNA prispevajo k preoblikovanju genoma in evoluciji. Zaporedja, ki so zaradi njih nastala, zajemajo 45 % človeškega genoma. Pri človeku je edini aktivni mobilni element LINE-1, ki ga uvrščamo med retrotranspozone brez dolgih končnih ponovitev. Retrotranspozoni LINE-1 so zmožni avtonomne premestitve, prav tako sodelujejo tudi pri neavtonomnih premestitvah drugih. Njihovo zaporedje vsebuje dva delujoča smerna bralna okvirja, ki zapisujeta proteina ORF1p, ORF2p in protismerni zapis za ORF0. Protein ORF1p se veže na RNA in deluje kot šaperon. Protein ORF2p ima endonukleazno in reverznotranskriptazno aktivnost. Le skupaj omogočata uspešno retrotranspozicijo, saj v primeru mutacije ali utišanja enega ta ne poteče. Premestitve LINE-1 v genomu povzročajo genetsko nestabilnost in so vzrok za veliko bolezni, med drugimi tudi za nevrodegenerativna obolenja. Da bi odkrili nove, učinkovitejše načine zdravljenja, sta ključna razumevanje molekularnega procesa retrotranspozicije in poznavanje molekul, ki v procesu sodelujejo, ter drugih, ki z njimi interagirajo. V okviru diplomske naloge smo določali vpliv proteina TDP-43 na retrotranspozicijo LINE-1. Protein TDP-43 je povezan z nevrodegenerativnimi boleznimi, saj njegovo kopičenje v citoplazmi v obliki agregatov povzroči smrt nevronov. V sesalskih celičnih linijah HEK293T smo spremljali retrotranspozicijo LINE-1 z vektorjem, ki ob uspešni retrotranspoziciji omogoča sintezo zelenega fluoroscenčnega proteina. Število zelenih celic smo prešteli s pretočnim citometrom. Retrotranspozicija je potekla povprečno v 10 % celic. S tehnikami prenosa western ter imunodetekcije smo dokazali uspešnost transfekcije. Z mikroskopijo smo določili, da je ta bila 93-%. Celice HEK293T smo transficirali tudi z vektorjem z zapisom za človeški protein TDP-43. V prvem eksperimentu je bilo zmanjšanje deleža retrotranspozicije LINE-1 v celicah HEK293T v odvisnosti od TDP-43 29-%. Ko smo izvedli še drugo tehnično ponovitev, rezultati niso bili statistično značilni. Za zanesljiv rezultat bi morali izvesti še vsaj eno biološko ponovitev poskusa.

• Language: Slovenian
• Keywords: retrotranspozicija, LINE-1, TDP-43, HEK293T, Flp-In 293
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• Publication date in RUL: 05.09.2024

Secondary language

• Language: English
• Title: Effects of TDP-43 on retrotransposition of LINE-1 in mammalian cell cultures

Abstract

Mobile DNA elements contribute to genome remodelling and evolution. Sequences they formed comprise 45% of the human genome. In humans, the only active mobile element is LINE-1, which is classified as a retrotransposon without long terminal repeats, they are transpositionally active. LINE-1 retrotransposons are capable of autonomous translocation and participate in non-autonomous translocations of others. Their sequence is written by two functional directional reading frames, which encode the proteins ORF1p, ORF2p and the antisense script for ORF0. The ORF1p protein binds to RNA and acts as a chaperone. The ORF2p protein has endonuclease and reverse transcriptase activity. Only together they enable successful retrotransposition. In the case where one is either mutated or silenced, they are incapable of effective retrotransposition. LINE-1 translocations in the genome cause genetic instability and are the cause of many diseases, including neurodegenerative diseases. To discover new, more effective therapies, it is crucial to understand the molecular process of retrotransposition, to know the molecules that participate in the process and others that interact with them. In this diploma, we determined the influence of the TDP-43 protein on LINE-1 retrotransposition. The TDP-43 protein is associated with neurodegenerative diseases, as its accumulation in the cytoplasm in the form of aggregates causes the death of neurons. Retrotransposition of LINE-1 was monitored in HEK293T mammalian cell lines with a vector that synthesizes green fluorescent protein upon successful retrotransposition. The number of green cells was counted using a flow cytometer. Retrotransposition took place in 10% of cells on average. Using western blotting and immunodetection techniques, we demonstrated the success of the transfection, with microscopy we determined it has occurred in 93% of the cells. HEK293T cells were also transfected with a human TDP-43 expression vector. In the first experiment, the TDP-43-dependent decrease in the proportion of LINE-1 retrotransposition in HEK293T cells was 29%. When we performed a second technical repetition, the results were not statistically significant. For a reliable result, at least one more biological repetition of the experiment should be carried out.

• Keywords: retrotransposition, LINE-1, TDP-43, HEK293T, Flp-In 293