20.500.12556/RUL-162816 Transport retrotranspozonov LINE1 v jedro s karioferini Transport of LINE1 retrotransposons into nucleus by karyopherins Retrotranspozoni LINE1 (ang. long interspersed nuclear element) so avtonomni mobilni DNA elementi, ki predstavljajo 17 % človeškega genoma in so edina aktivna različica transpozicijskih elementov pri človeku. Gen LINE1 vsebuje zapis za tri proteine: ORF0p, ORF1p in ORF2p. Slednja sta glavna gradnika pri sestavi ribonukleoproteinskega kompleksa, povezuje pa ju lastna mRNA. Kompleks je pomemben za aktivno retrotranspozicijo in vnos lastne DNA na novo mesto v genomu. Prenos kompleksa v jedro ni povsem znan, potrjeno pa ta potuje med celično delitvijo, ko se zmanjša integriteta jedrni ovojnici. Prisotnost retrotranspozona LINE1 je bila potrjena tudi pri somatskih nevronskih celicah, kar pomeni, da se lahko prenese tudi preko jedrne ovojnice. En izmed potencialnih načinov je z jedrnimi importini, imenovani tudi karioferini. Transportni kompleks je sestavljen iz tovora, karioferina α, ki prepozna tovor, in karioferina β1, ki je odgovoren za vezavo na karioferin α in prenos skozi jedrno poro. V prvem delu magistrske naloge smo izolirali proteine, ki so potrebni za izvedbo testa jedrnega uvoza. Izolirali smo proteine ORF1p, vseh sedem karioferinov α in karioferin β1, povezane s fuzijskim proteinom GST. Vsi proteini so se nahajali v topni obliki in smo jih uspešno očistili. V drugem delu smo želeli preveriti, če obstajajo interakcije med ORF1p in karioferini α. V ta namen smo s koimunoprecipitacijo preverili, če se prekomerno izražen ORF1p-FLAG poveže z endogenimi karioferini α. Z rezultati nismo potrdili medsebojnega interagiranja med ORF1p in karioferini α. V tretjem delu smo testirali posamezne karioferine α kot potencialne prenašalce ORF1p v jedro. Pri testu jedrnega uvoza (NIA) smo sesalskim celicam HeLa odstranili citoplazmo in dodali samo ORF1p ter izbran karioferin α in β1. S protitelesi smo lokalizirali posamezne proteine in pokazali, da sta karioferina α3 in α4 jedrna prenašalca ORF1p. Pri testu NIA se je ORF1p pred dodatkom prenašalcev nahajal izven jedra, z dodatkom karioferinov α3 in α4 pa večinoma v jedru. Pomemben dejavnik pri testu je bil tudi dodatek energije v obliki ATP, ki je bil ključen za uspešen prenos. Z inhibitorjem jedrnih por WGA smo dokazali, da je prenos ORF1p možen skozi jedrne pore tudi pri nedelečih se celicah. V prihodnjih raziskavah bi bilo potrebno preveriti interakcije med izoliranimi proteini in optimizirati test jedrnega uvoza. LINE1 retrotransposons are autonomous mobile DNA elements that represent 17% of the human genome and are the only active variant of transposition elements in human. The LINE1 gene contains the transcript for three proteins: ORF0p, ORF1p and ORF2p. The latter two are the main building elements in the assembly of the ribonucleoprotein complex, and they are connected by their own mRNA. The complex is important for active retrotransposition and introduction of their own DNA to a new place on the genome. The transfer of the complex to the nucleus is not completely known, but it has been confirmed that it travels during cell division, when the integrity of the nuclear envelope is reduced. The presence of the LINE1 retrotransposon was also confirmed in somatic neuronal cells, which means that it can also be transferred through the nuclear envelope. One potential way is with nuclear importins, called karyopherins. The transport complex consists of the cargo, karyopherin α, which recognizes the cargo, and karyopherin β1, which is responsible for binding to karyopherin α and transport through the nuclear pore. In the first part of the master's thesis, we isolated the proteins that are necessary to perform the nuclear import assay (NIA). We isolated ORF1p, all seven karyopherins α, and karyopherin β1 with GST-tag. In the second part, we wanted to check the interactions if there are any between ORF1p and karyopherins α. For this purpose, we checked with co-immunoprecipitation whether overexpressed ORF1p-FLAG binds to endogenous karyopherins α. The results did not confirm a mutual interaction between ORF1p and karyopherins α. In the third part, we tested individual karyopherins α as potential ORF1p transporters to nucleus. In the nuclear transport assay, we washed cytoplasm from mammalian HeLa cells. Only ORF1p and selected karyopherin α and β1 were added. Individual proteins were localized with antibodies and mutual colocalization showed that karyopherins α3 and α4 are nuclear transporters of ORF1p. ORF1p was located outside the nucleus before NIA assay, but with addition of karyopherins α3 and α4 was mostly found in the nucleus. An important factor in the test was also the addition of energy as ATP, which was crucial for a successful transfer. With nuclear pore inhibitor WGA, we proved that the transfer of ORF1p through the nuclear pores is possible even in non-dividing cells. In future research, it would be necessary to retrotranspozon LINE1 ORF1p karioferin α test jedrnega uvoza koimunoprecipitacija retrotransoposon LINE1 ORF1p karyopherin α nuclear import assay coimmunoprecipitation true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-09-27 13:05:03 2024-09-27 13:05:14 2024-11-28 10:39:42 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 22884 216817667 Nograsek_Tim_-_Transport_retrotranspozonov_LINE1_v_jedro_s_karioferini.pdf Nograsek_Tim_-_Transport_retrotranspozonov_LINE1_v_jedro_s_karioferini.pdf 1 CB8C35E00F028E8926C4FEDD5B85D7D3 7e446bcf4bdb392da7f0a1387b0cd8b2351f512f8441639d61a24323baeecb2c 40dd2e76-7cc0-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=192249 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0