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Determination of proteins bound to dipeptide repeats
Get PDFCelični mehanizmi, odgovorni za nastanek patologije, ki vodi v amiotrofično lateralno sklerozo, še niso dobro poznani in trenutno ni zdravil, ki bi bolezen pozdravila. Proteini z dipeptidnimi ponovitvami (DPR), ki se prevajajo iz podaljšanih heksanukleotidnih ponovitev znotraj gena C9orf72, imajo sposobnost vezave na različne znotrajcelične proteine. Le-ti pri bolnikih z amiotrofično lateralno sklerozo nastanejo pri posebni obliki translacije, ki poteka neodvisno od začetnega kodona AUG. V nalogi smo izražali pet različnih zvrsti dipeptidnih ponovitev (poli (GA), poli (GP), poli (GR), poli (PA) in poli (PR)), ki smo jih konjugirali z encimom BioID2, in z metodo BioID poiskali kandidatne celične proteine, ki se prehodno ali stalno vežejo na DPR, ali pa se nahajajo v njihovi bližini. Z ontološkimi analizami kandidatnih proteinov smo določili, da so proteini poli (GA) vpleteni v procese, povezane z mikrotubuli, in v katabolične procese proteinovproteini poli (GP) vpleteni v procese prenosa signalaproteini poli (GR) vpleteni v procese začetka prevajanja proteinov in metabolične procese amidovproteini poli (PA) vpleteni v uravnavanje encimske aktivnosti in pozitivnega uravnavanja apoptozeproteini poli (PR), vpleteni v procesiranje rRNA. Z magistrsko nalogo smo prispevali nova spoznanja o celičnih mehanizmih pri najpogostejši mutaciji amiotrofične lateralne skleroze, ki bodo, skupaj z nadaljnjimi raziskavami, pripomogla k boljšemu razumevanju bolezni in novim načinom zdravljenja ali preprečevanja bolezni.The cellular mechanisms that are responsible for the development of pathology leading to amyotrophic lateral sclerosis are not yet well understood, and currently there is no cure for the disease. Dipeptide repeat (DPR) proteins that are translated from extended hexanucleotide repeats within the C9orf72 gene can bind to various intracellular proteins. These proteins in patients with amyotrophic lateral sclerosis are generated by a special form of repeat-associated non-AUG translation. We expressed five different types of dipeptide repeats (poly(GA), poly(GP), poly(GR), poly(PA) and poly(PR)), which were conjugated with the BioID2 enzyme, and with the BioID method determined candidate intracellular proteins that transiently or permanently bind to DPRs or can be found in their vicinity. With ontological analysis of candidate proteins we determined that poly(GA) proteins are involved in microtubule-based processes and in protein catabolic processespoly(GP) proteins are involved in signal transduction processespoly(GR) proteins are involved in translational initiation processes and in cellular amide processespoly (PA) proteins are involved in the regulation of catalytic activity and in positive regulation of apoptotic processespoly (PR) proteins are involved in the rRNA processing. With this master thesis we have contributed new findings about cellular mechanisms in the most common mutation of amyotrophic lateral sclerosis, which will along with further research contribute to a better understanding of the disease as well as new ways of treatment and prevention of the disease
Impact of genes linked to amyotrophic lateral sclerosis on biochemical properties and cellular localization of the TDP-43 protein
Get PDFUvod: Amiotrofična lateralna skleroza (ALS) je smrtonosna napredujoča nevrodegenerativna bolezen, za katero so značilni citoplazemski skupki proteinov v prizadetih nevronih. Glavna komponenta teh inkluzij je ubikvitiniran, fosforiliran in cepljen protein TDP-43. Primerov ALS z mutacijami v genu je TDP-43 zelo malo, kar pomeni, da ima TDP-43 v patoloških agregatih pri večini pacientov povsem običajno zaporedje. Vedno več raziskav nakazuje, da imajo citoplazemski skupki TDP-43 lastnosti podobne prionskim proteinom. Zanje je značilno sejanje agregatov in njihovo širjenje med celicami in vitro in in vivo, novejše študije pa so identificirale tudi različne tipe agregatov pri bolnikih z ALS in frontotemporalno demenco (FTD). Alternativne patološke konformacije agregatov bi lahko podobno kot sevi prionskih proteinov, bile osnova za raznolikost proteinopatij TDP-43 in heterogenost bolezni. Raziskovalne skupine so poleg TDP-43 identificirale že več kot 50 genov, ki povzročajo ali vplivajo na nastanek ALS in so vključeni v dve skupini celičnih procesov: metabolizem RNA in razgradnja proteinov. Pri tem pa vloga drugih genov, povezanih z ALS in njihovih mutacij na agregacijo TDP-43, ostaja neznanka.
Namen dela in hipotezi: V pričujoči študiji smo primerjali vpliv več mutiranih in nemutiranih genov, povezanih z ALS, na agregacijo TDP-43 in biokemijske lastnosti agregatov in vitro. Hipotezi, na katerih sloni raziskava, se osredotočata na dva vidika agregacije proteina TDP-43 - na vpliv genov povezanih z ALS na agregacijo TDP-43 in na citoplazemsko lokalizacijo agregatov TDP-43: Izražanje z ALS povezanih genov (UBQLN2, MATR3, VCP, hnRNPA1) vpliva na biokemijske lastnosti in/ali celično lokalizacijo proteina TDP-43. Modeliranje okvar jedrno-citoplazemskega transporta v celicah zaostri patološke spremembe, ki so posledica izražanja posamezne mutacije povezane z ALS.
Metode dela: Da bi raziskali oba vidika nastanka agregatov TDP-43 smo najprej razvili nov in vitro model agregacije TDP-43 v celični liniji SH-SY5Y. Okvare jedrno-citoplazemskega transporta TDP-43 smo modelirali z eliminacijo signala za jedrno lokalizacijo (NLS) in s tem premaknili TDP-43 iz jedra v citoplazmo. Nato smo postopoma skrajševali LCD proteina in s tem zmotili fiziološko konformacijo dimerov TDP-43. Pri ko-transfekcijskih eksperimentih, s katerimi smo želeli preveriti vpliv prej naštetih mutiranih in nemutiranih z ALS-povezanih genov na agregacijo TDP-43, smo uporabili celoten TDP-43 z odstranjenim NLS (dNLS) in TDP-43 brez NLS in IDR2 (dNLSd343). Nastanek agregatov v kotransfeciranih celicah smo kvantificirali s konfokalno mikroskopijo in nadalje analizirali s programom ImageJ z dodanim vtičnikom Shape Descriptors. Topnost oziroma netopnost agregatov v pufrih RIPA in UREA smo analizirali s prenosom western.
Rezultati in razprava: Pokazali smo, da krajšanje domene LCD vodi do različnih vzorcev agregacije TDP-43. S ko-transfekcijo konstruktov dNLS in dNLSd343 z mutiranimi in nemutiranimi z ALS-povezanimi geni smo pokazali, da ima vsak izmed testiranih genov edinstven vpliv na vzorec agregacije TDP-43 in na topnost agregatov. Te učinke smo preučevali v kontekstu okvarjenega nukleocitoplazemskega transporta, ki je zaostril patološko agregacijo v celicah ko-transfeciranih z mutiranimi ali nemutiranimi z ALS-povezanimi geni. Nekateri geni, kot sta na primer mutirani in nemutirani hnRNPA1 so zmanjšali število agregatov in povečali njihovo topnost, drugi pa so povečali število agregatov in/ali njihovo velikost in/ali zmanjšali njihovo topnost. Pokazali smo tudi, da vsak izmed genov edinstveno vpliva tako na dNLS kot na dNLSd343, kar kaže na pomembno vlogo skrajnega C-končnega dela domene LCD pri nastanku in maturaciji agregatov. Izsledki naše študije tako potrjujejo, da vsak izmed z ALS-povezanih genov, ki smo jih raziskovali, edinstveno vpliva na procese agregacije TDP-43, kar vodi do nastanka različnih »tipov agregatov,« ki bi lahko predstavljali temelj za heterogenost bolezni ALS.
Zaključki in znanstveno-raziskovalni pomen študije: Študija, predstavljena v tej doktorski disertaciji je prva, ki primerja vpliv z ALS-povezanih genov na agregacijo proteina TDP-43. V sklopu študije smo tudi razvili model agregacije proteina TDP-43, s katerim bi lahko v prihodnosti raziskovali zmožnost potencialnih terapevtskih učinkovin za zmanjšanje ali eliminacijo agregatov TDP-43 in s tem izboljšanje patoloških sprememb v prizadetih celicah bolnikov z ALS. Naši rezultati potrjujejo potrebo po primerjalnih raziskavah na področju ALS in razkrivajo, kako različni mehanizmi interagirajo na molekulskem nivoju in vodijo v nevrodegeneracijo, kar se bo morda izkazalo kot ključnega pomena za uspešen razvoj terapevtikov.Introduction: ALS is a fatal progressive neurodegenerative disease pathologically characterized by cytoplasmic deposits of misfolded proteins in the affected neurons. The main component of these inclusions is ubiquitinated, phosphorylated and cleaved TDP-43 protein. Mutated TDP-43 is very rare, meaning that most of the patients with TDP-43 positive aggregates do not carry any mutation in this protein. Mounting evidence suggests that cytoplasmic accumulations of TDP-43 exhibit prion-like characteristics. In addition to seeding and intercellular propagation of TDP-43 aggregation between cells in vitro and in vivo, different types of the TDP-43 aggregates in ALS and FTD diseased brains have been identified. It has been proposed, that alternate pathological conformations may form the basis for the diversity of TDP-43 proteinopathies and disease heterogeneity, reminiscent of prion strains. So far, more than 50 potentially causative or ALS-modifying genes have been identified, mainly involved in two cellular processes: RNA metabolism and quality control of protein metabolism. However, the role of ALS-associated genes, the possible impact of their mutations on TDP-43 aggregate behavior and properties remains largely unknown.
Aim and Hypotheses: In this study, we sought to compare the impact of several wild-type and mutated ALS-associated genes on TDP-43 aggregation in vitro. Our hypotheses focused on two aspects of TDP-43 aggregation. On the impact, ALS-associated genes display on TDP-43 aggregation and on the cytoplasmic localization of TDP-43 aggregates. Expression of genes (UBQLN2, MATR3, VCP and hnRNPA1) linked to ALS affects biochemical properties and/or cellular localization of protein TDP-43. Nucleo-cytoplasmic transport defect modelling exacerbates pathological changes resulting from the expression of a certain ALS mutation.
Methods: To explore both aspects of TDP-43 aggregation, we first developed a novel in vitro TDP-43 aggregation model in neuroblastoma SH-SY5Y cell line. To model defects of nucleo-cytoplasmic transport and achieve TDP-43 cytoplasmic localization, we eliminated NLS from the full-length TDP-43 sequence. Then we stepwise shortened its LCD, to disrupt physiological conformation of TDP-43 dimers. Full-length TDP-43 lacking only NLS (dNLS) and TDP-43 without NLS and IDR2 (dNLSd343) constructs were used in co-transfection experiments to assess the impact of pre-selected wild-type and mutant ALS-associated genes on TDP-43 aggregate behavior. Aggregate formation in co-transfected cells was quantified by confocal microscopy and further analyzed by ImageJ software with Shape Descriptors plugin. Solubility and insolubility of the aggregates in UREA and RIPA buffers was analyzed by western blotting.
Results and Discussion: We demonstrated that shortening of the LCD domain leads to distinct patterns of TDP-43 aggregation. By co-transfecting dNLS and dNLSd343 constructs with wild-type and mutant ALS-associated genes, we demonstrated that each of the tested genes has a unique influence on TDP-43 aggregation pattern and solubility of the aggregates. These effects were studied in the context of disrupted nucleocytoplasmic transport, which caused exacerbation of pathological aggregation following co-transfection with wild-type or mutant ALS-associated genes. While some of the ALS-associated genes, such as wt and mut hnRNPA1 reduced the number of aggregates and increased their solubility, others increased the number of aggregates and/or their size and/or decreased their solubility. We also show that each of the genes has a unique influence on both dNLS and dNLSd343, suggesting that the extreme C-terminus has a prominent role in initiation and maturation of the aggregates. Considering all this data, we propose that each ALS-linked gene we studied uniquely affects TDP-43 aggregation processes, leading to the formation of distinct “aggregate types,” which could represent the basis for the disease heterogeneity.
Conclusions: The study presented in this doctoral dissertation is first to provide an insight into the impact ALS-associated genes have on TDP-43 aggregation. Additionally, as a part of our research, we developed a TDP-43 aggregation model, which could be used in future studies of therapeutic compounds with a potential to reduce or eliminate TDP-43 aggregates and thus reverse pathological changes in the affected cells of ALS patients. Our results also emphasize the significance of comparative research in the ALS field and provide an understanding on how different mechanisms interact at the molecular level and lead to neurodegeneration that may reveal crucial in the future for successful development of therapeutics
Detection of SNORD116 RNA targets using the COMRADES method
No full textVelika večina RNA molekul je nekodirajočih in imajo drugačne, a pomembne funkcije. Med te spadajo tudi snoRNA, ki so majhne, nekodirajoče RNA, ki se kopičijo v jedrcu in usmerjajo posttranskripcijske modifikacije mnogih drugih RNA molekul. Glede na značilna ohranjena zaporedja snoRNA delimo v dve podskupini, in sicer SNORD in SNORA. V tej nalogi smo se osredotočili na molekule SNORD oz. male nukleolarne RNA s C/D ohranjenim zaporedjem. Velika večina teh RNA vodi 2\u27 O metilacijo ribosomske RNA, obstajajo pa tudi take, ki nimajo znanih funkcij oz. tarč, s katerimi se povezujejo, zato jih imenujemo tudi snoRNA sirote. Med slednje sodi tudi genska gruča SNORD116, ki je opredeljena kot minimalna kritična regija Prader Willijevega sindroma (PWS). To je redka genetska nevrorazvojna motnja, ki je posledica delecije po očetu podedovanih genov na kromosomu 15 in lahko med drugim povzroči življenjsko ogrožajočo debelost. Ker je genska gruča SNORD116 odsotna pri vseh bolnikih s PWS, velja za glavni genetski dejavnik za pojav sindroma.
V magistrski nalogi smo optimizirali protokol metode COMRADES, s katero smo nato želeli identificirati RNA tarče SNORD116. Metoda temelji na in vivo prečnem povezovanju RNA dupleksov s psoralen-TEG azidom, ki ob obsevanju z dolgovalovno UV svetlobo tvori kovalentne povezave med dvema verigama RNA. Temu sledi izolacija celokupne RNA iz celic in biotinilacija prečno povezanih dupleksov, ki jih nato afinitetno zajamemo s testom »pull down«. Prečno povezane fragmente ločimo s poliakrilamidno gelsko elektroforezo, nato pa z bližnjo ligacijo povežemo konca obeh prečno povezanih molekul in z obsevanjem s kratkovalovno UV svetlobo prekinemo kovalentne povezave med parom RNA verig. Nastanejo hibridne enoverižne RNA molekule, iz katerih sintetiziramo knjižnico cDNA in s sekvenciranjem ter bioinformatsko analizo poskušamo identificirati pare SNORD116 in njenih tarčnih RNA.
Uspešno smo optimizirali več korakov protokola ter dodali korak obogatitve izbrane RNA, kjer s hibridizacijo z biotiniliranimi DNA-sondami in tehniko afinitetnega zajema povečamo delež SNORD116 v vzorcu in se izognemo prevelikim izgubam. Zaradi obsežnega eksperimentalnega dela naloge na koncu nismo uspeli identificirati RNA tarč SNORD116. Kljub temu bo optimiziran protokol služil kot temelj za prihodnje eksperimente, ki bodo poskušali razkriti funkcije te enigmatične genske gruče.The vast majority of RNA molecules are non-coding and have different but important functions. Among these are snoRNAs, which are small non-coding RNAs that accumulate in the nucleus and direct post-transcriptional modifications of many other RNA molecules. Based on the characteristic conserved sequences, snoRNAs are divided into two subgroups, SNORD and SNORA. In this thesis, we focused on SNORD molecules or C/D box snoRNAs. The majority of these RNAs guide 2\u27 O methylation of ribosomal RNA, but there are also those that have unknown functions or targets, and they are referred to as snoRNA orphans. The group includes SNORD116 gene cluster, which is defined as the minimal critical region for Prader-Willi syndrome (PWS). PWS is a rare genetic neurodevelopmental disorder caused by a paternal deletion of genes on chromosome 15 and can lead to life-threatening obesity, among other symptoms. Since the SNORD116 gene cluster is absent in all PWS patients, it is considered a key genetic factor in the syndrome’s development.
In this master\u27s thesis, we optimized the COMRADES method protocol to identify RNA targets of SNORD116. The method is based on in vivo cross-linking of RNA duplexes with psoralen-TEG azide, forming covalent bonds between two RNA strands upon long wavelength UV irradiation. This process is followed by the isolation of total RNA from cells, partial fragmentation, and biotinylation of cross-linked duplexes, which are then affinity captured using a "pull down" assay. Cross-linked fragments are separated by polyacrylamide gel electrophoresis, after which we ligate the ends of both cross-linked molecules and use short-wavelength UV light to break the covalent bonds between the RNA strand pair. This results in hybrid single-stranded RNA molecules from which we synthesize a cDNA library and attempt to identify pairs of SNORD116 and its target RNAs through sequencing and bioinformatic analysis.
We successfully optimized several protocol steps and added a step to enrich the selected RNA. By hybridizing with biotinylated DNA probes and using affinity capture techniques, we increase the relative amount of SNORD116 in the sample and avoid significant losses. However, due to the extensive experimental work in this thesis, we were not able to identify the RNA targets of SNORD116 in the end. Nevertheless, the optimized protocol will serve as a foundation for future experiments aiming to uncover the functions of this enigmatic gene cluster
Role of antisense RNA transcript from C9orf72 mutation associated with amyotrophic lateral sclerosis and frontotemporal dementia
No full textUvod: Amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD) sta hitro napredujoči, neozdravljivi in smrtonosni nevrodegenerativni bolezni. Najpogostejši genetski vzrok ALS in FTD je mutacija v genu C9orf72 (angl. chromosome 9 open reading frame 72). Mutacija se kaže v obliki razširjenih ponovitev zapisa G4C2, katerih število pri obolenju doseže več 100 lahko tudi več 1000 ponovitev. Pri ALS in FTD naj bi mutacija povzročala: haploinsuficienco proteina C9orf72, z RNA posredovano toksičnost smernih in protismernih prepisov RNA razširjenih ponovitev ter toksičnost proteinov z dipeptidnimi ponovitvami (DPR), ki se prevedejo iz smernih in protismernih zapisov RNA. Z RNA posredovana toksičnost je posledica sekvestracije proteinov na skupke smernih in protismernih zapisov RNA, kar onemogoči njihove normalne funkcije v celici. Obstaja veliko raziskav o vezavi različnih proteinov na smerne zapise RNA. Vezava proteinov na protismerne zapise je raziskana v veliko manjši meri. Veliko raziskav je usmerjenih tudi v preučevanje DPR-jev in z njimi povezanih okvar celičnih procesov. Vse več pozornosti se namenja tudi povezavi med vsemi tremi mehanizmi, ki jih povzroča mutacija v genu C9orf72 ter na njihovo skupno vlogo v patologiji bolezni.
Namen dela in hipoteza: Tako smerni kot protismerni zapisi RNA razširjenih ponovitev G4C2 v genu C9orf72 vežejo nase različne proteine, kar prispeva k patologiji ALS in FTD. Ker so v večji meri raziskani le smerni zapisi RNA smo se v naši raziskavi osredotočili na manj raziskane protismerne zapise RNA ponovitev v genu C9orf72. Hipoteza, na kateri sloni raziskava, se glasi: Protismerni zapis RNA mutacije v genu C9orf72 veže za celico pomembne proteine, kar je lahko povezano s patologijo ALS in FTD. Namen dela je bil identificirati proteine, ki vežejo protismerni zapis RNA razširjenih ponovitev mutacije v genu C9orf72 ter opredeliti potencialno vlogo teh interakcij pri patologiji ALS in FTD. Poleg tega smo v nalogi ovrednotili vpliv proteina SFPQ (angl. splicing factor proline and glutamine rich), za katerega je znano, da veže smerne zapise RNA ponovitev v genu C9orf72, na število jedrnih skupkov tako smernih kot protismernih zapisov RNA ponovitev v genu C9orf72 ter na raven izražanja DPR-jev.
Metode dela: Za identifikacijo proteinov, ki vežejo dolge ponovitve protismerne RNA iz razširjenih ponovitev G4C2 v genu C9orf72, smo vzpostavili test RNA “pull down”. Interakcijo protismerne RNA z identificiranimi proteini smo preverili na celicah s prisotno mutacijo v genu C9orf72 s pomočjo RNA fluorescenčne in situ hibridizacije (RNA-FISH) v kombinaciji z imunocitokemijo (ICC) in analizo kolokalizacije fluorescenčnega signala skupkov RNA in izbranih proteinov. Kot prvi smo optimizirali metodo RNA-protein PLA (angl. proximity ligation assay) za analizo interakcij protismernih ponovitev RNA in proteinov v citoplazmi tudi izven znanih jedrnih skupkov RNA. Optimizirali smo protokol za preverjanje stopnje aminoacilacije tRNA v kombinaciji s kvantitativno verižno reakcijo s polimerazo (qPCR) in naše rezultate uspešno analizirali z metodo ddCt. Vpliv ravni izražanja proteina SFPQ na število skupkov RNA in na izražanje DPR-jev v celicah smo ovrednotili v vzpostavljenih celičnih modelih s prekomerno izraženim ali utišanim proteinom SFPQ. V teh celičnih modelih smo izražali smerne oziroma protismerne zapise razširjenih ponovitev gena C9orf72. Za prekomerno izražanje proteina SFPQ smo uporabili transfekcijo, za njegovo utišanje pa transdukcijo celic z lentivirusnimi delci. Raven izražanja A podenote fenilalanin-tRNA sintetaze (FARSA), SFPQ in DPR-jev smo preverili s prenosom western in točkovnim nanosom vzorcev z analizo intenzitete končnega protitelo-antigen signala. Detekcijo posameznih skupkov RNA in proteinov ter signala RNA-protein z metodo PLA smo izvedli s konfokalno fluorescenčno mikroskopijo. Kvantifikacija je bila izvedena s programom ImageJ. Za statistično analizo smo uporabili Studentov t-test.
Rezultati in razprava: Pokazali smo, da C4G2 ponovitve RNA vežejo proteine vpletene v različne celične procese. Identificirani proteini sodelujejo pri sestavi in stabilnosti citoskeleta, transportu RNA in proteinov po aksonih in dendritih (CYFIP1/2, TAOK1), pri procesiranju in transportu molekul RNA (HNRNPL), pri sintezi proteinov (FARSA/B), pri funkciji mielinizirajočih celic (CNP), pri biogenezi ribosomov in pri preverjanju kvalitete proteinov (NPM1). Identificirani proteini so bili že povezani z različnimi nevrodegenerativnimi boleznimi, med njimi tudi z ALS. V nadaljevanju raziskave smo se osredotočili na citoplazemski interaktor fenilalanin-tRNA sintetazo (FARS), ki je sestavljena iz podenote ? (FARSA), katere naloga je vezava ustrezne aminokisline na tRNA in podenote ß (FARSB), katere naloga je odstranitev nepravilno pripete aminokisline iz tRNA. Z namenom preučevanja citoplazemskih interakcij protismerne RNA C4G2 s proteini v celičnih modelih, smo optimizirali protokol RNA-protein PLA detekcije. Interakcije RNA C4G2 s FARSA smo uspeli potrditi v treh različnih celičnih linijah z mutacijo v genu C9orf72 (fibroblastih, limfoblastih in iPSC-ih). Okvare v
delovanju FARSA povzročene z vezavo na C4G2 RNA bi lahko vodile v znižano aminoacilacijo Phe-tRNA. Zato smo optimizirali protokol za določanje stopnje aminoacilacije tRNA, s katerim smo v limfoblastih s prisotno mutacijo v genu C9orf72 odkrili bistveno znižanje ravni aminoacilacije Phe-tRNA, v primerjavi s kontrolnimi limfoblasti. Napake v delovanju aminoacil-tRNA sintetaz (ARS) lahko vodijo v napačno zvijanje in kopičenje proteinov, kar je značilno za nevrodegenerativne bolezni. Poleg tega imajo ARS-i še veliko nestandardnih funkcij, ki bi bile lahko prav tako prizadete ob interakciji teh proteinov s ponovitvami C4G2 RNA v celicah. V drugem delu naloge smo proučili vpliv proteina SFPQ, povezanega z nevrodegeneracijo, na tvorbo skupkov smerne in protismerne RNA razširjenih ponovitev gena C9orf72 in na sintezo DPR-jev. Znižana raven izražanja proteina SFPQ zniža število skupkov smerne in v manjši meri tudi protismerne RNA v celici, ter zniža raven izražanja DPR-jev. Prekomerno izražanje proteina SFPQ pa poveča število skupkov smerne in protismerne RNA ter sintezo DPR-jev. SFPQ je znan interaktor smerne RNA razširjenih ponovitev gena C9orf72, mi pa smo pokazali, da ne veže protismerne RNA razširjenih ponovitev gena C9orf72. S tem lahko razložimo manjši vpliv ravni izražanja SFPQ na skupke protismerne RNA. Pokazano je bilo, da protein SFPQ uravnava prepisovanje genov, ki imajo kompleksno sekundarno strukturo na ravni DNA. Predvidevamo, da posledično lahko vpliva tudi na prepisovanje razširjenih ponovitev gena C9orf72, ki prav tako tvorijo sekundarne strukture na ravni DNA. To pojasni vpliv ravni izražanja proteina SFPQ na število tako smernih kot protismernih skupkov RNA ponovitev C9orf72 ter tudi na sintezo DPR-jev, ki smo jo opazili. Spremenjena sinteza DPR-jev pa bi bila lahko uravnavana tudi na ravni translacije, saj je SFPQ vpleten tudi v mehanizme translacije.
Zaključki in znanstveno-raziskovalni pomen študije: Določili smo nove proteine, ki vežejo manj raziskane protismerne zapise RNA razširjenih ponovitev gena C9orf72, ki so vpleteni v različne celične procese. Okvare funkcije teh proteinov zaradi njihove vezave na protismerno RNA bi lahko imele značilne posledice za delovanje nevronov. Prvi smo odkrili interakcijo proteina FARS s protismernimi RNA razširjenih ponovitev gena C9orf72 in okvaro aminoacilacije tRNA v celicah z mutacijo v genu C9orf72 in tako pomembno prispevali k razumevanju okvare mehanizmov in prizadetosti celičnih procesov pri ALS in FTD s prisotno mutacijo v genu C9orf72. Odkriti mehanizem odpira nove možnosti za terapevtske pristope pri ALS in FTD obolenjih. Za ovrednotenje
citoplazemske interakcije protismerne RNA, smo prvi v ta namen optimizirali in uporabili metodo RNA-protein PLA. Do sedaj so metode omogočale le analize interakcij razširjenih ponovitev RNA znotraj jedra. Z našo optimizirano RNA-protein PLA metodo pa smo omogočili tudi analize interakcij v citoplazmi. S tem smo pomembno prispevali k metodološkem pristopu analize interakcij razširjenih ponovitev RNA mutacije v genu C9orf72 z različnimi celičnimi proteini. Prav tako smo pokazali, da raven izražanja proteina SFPQ vpliva na tvorbo skupkov tako smerne kot protismerne RNA in na sintezo DPR-jev. S tem smo pripevali k razumevanju vloge proteina SFPQ pri bolezni ALS in FTD in njegovega vpliva na toksične mehanizme, ki jih mutacija v genu C9orf72 proži. Uravnavanje ravni SFPQ-ja in z njim povezanih mehanizmov je potencialen terapevtski pristop za zdravljenje ALS in FTD s prisotno mutacijo v genu C9orf72.Introduction: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fast progressive and fatal neurodegenerative diseases. The most common genetic cause of both diseases is mutation in the chromosome 9 open reading frame 72 gene (C9orf72). The mutation is expanded repeat of G4C2 sequence. In disease the number of repeats reaches more hundreds and more thousand units. There are three proposed mechanisms of action for the mutation: haploinsufficiency of C9orf72 protein, RNA toxicity exerted by sense and antisense RNA repeats, and toxicity of dipeptide repeat proteins (DPRs) translated from the sense and antisense RNA repeats. RNA toxicity is the consequence of sequestration of RNA-binding proteins by sense and antisense RNA repeats, which impairs their normal cellular function. Many studies aimed to identify proteins binding to sense RNA repeatshowever, there is a distinct lack of studies of antisense RNA repeats. Another major subject of the studies related to the C9orf72 mutation are DPRs and their influence on cell mechanisms. The prevailing view is that all C9orf72 mutation mechanisms work together in disease pathology.
Aims and Hypothesis: Both sense and antisense RNA transcripts from C9orf72 mutation bind various proteins, which contributes to the pathology of ALS and FTD. This research is focused on the less studied antisense RNA. The study is based on the following hypothesis: Antisense RNA transcripts of C9orf72 gene mutation bind important cellular proteins and these interactions may be associated with pathology of ALS and FTD. Our aim was to identify proteins binding to antisense RNA repeats and define their potential role in disease pathology. Moreover, we also wanted to define the impact of known sense RNA interactor splicing factor proline and glutamine rich (SFPQ) on the number of sense and antisense RNA foci and DPR expression levels, as we propose it impacts stability and formation of RNA foci and, consequently, DPR production.
Methods: In order to identify the proteins binding to long antisense repeats, we set up RNA pull-down experiment. We studied the antisense RNA-protein colocalizations in C9orf72 mutation-positive patient-derived cells using RNA fluorescent in situ hybridization (RNA-FISH) in combination with immunocytochemistry (ICC) with overlap analysis of RNA foci and protein fluorescence signals. We were first to establish RNA-protein proximity ligation assay (PLA) for detection of interactions outside of nuclear RNA foci. To analyze the catalytic function of Phe-tRNA synthetase (FARS) in C9orf72 mutation, we optimized the tRNA aminoacylation assay in combination with quantitative polymerase chain reaction (qPCR). We analyzed the results with ddCt method. Furthermore, we studied the impact of SFPQ on sense and antisense RNA foci and DPRs by either SFPQ overexpression or knockdown in the cells expressing sense or antisense RNA repeats. Transfection was used for the protein overexpression and lentiviral transduction for the SFPQ knockdown in cells. The expression levels of FARSA, SFPQ, and DPRs were analyzed by western blot and dot blot. Densitometric analyses of the protein signals were performed. The fluorescently labeled RNA foci and proteins, and RNA-protein PLA signals in cells were analyzed by confocal microscopy. We used ImageJ for RNA foci and RNA-protein PLA signal counts. Statistical significance was calculated with the unpaired, two-tailed Student’s t-test.
Results and discussion: In this research, we identified various new proteins interacting with C4G2 RNA, which are implicated in multiple cellular mechanisms including composition and stability of cytoskeleton, axonal and dendritic transport (CYFIP1/2, TAOK1), RNA processing and transport (HNRNPL), protein synthesis (FARSA/B), myelinating cell functions (CNP), ribosome biogenesis and protein quality control (NPM1). The identified proteins have been previously associated with various neurodegenerative diseases, including ALS and FTD. We focused on the cytoplasmic interactor –FARS. FARS is composed of two subunits, FARSA is responsible for the attachment of cognate amino acid to its tRNA and FARSB has an editing function for elimination of amino acids from misacylated tRNAs. In order to study cytoplasmic interactions of antisense RNA with identified proteins, we optimized RNA-protein PLA method. We confirmed interactions of FARSA with antisense RNA in three C9orf72 mutation-positive patient-derived cell lines (fibroblasts, lymphoblasts and iPSCs) with RNA-protein PLA. Furthermore, in order to study the catalytic function of FARSA in C9orf72 mutation, we optimized the tRNA aminoacylation assay. We discovered there is a significant decrease of charged Phe-tRNA in C9orf72 mutation-positive patient-derived lymphoblasts compared to controls. Disruptions of aminoacyl-tRNA synthetases (ARS) have been previously shown to cause protein misfolding and accumulation, and are implicated in various disorders of neurological system. Moreover, ARSs have multiple uncanonical functions, which could also be affected and play role in multiple diseases. We also investigated the role of SFPQ in the formation of both sense and antisense RNA foci and the production of DPRs. SFPQ has been previously implicated in ALS and FTD and is a known interactor of sense RNA. Reduction in SFPQ expression levels led to reduction in the number of sense and antisense RNA foci and in the expression levels of DPRs. SFPQ overexpression increased the numbers of sense and antisense RNA foci and the DPRs production. Overall, the impact on antisense RNA foci was lower compared to sense RNA foci. We showed that, in contrast to sense RNA, SFPQ does not bind to antisense RNA, which could explain this effect. Nevertheless, the impact on numbers of antisense RNA foci was still significant, which could be the consequence of SFPQ involvement in transcription regulation of the C9orf72 expanded repeats. It was previously shown that SFPQ enables transcription of genes with complex secondary structures, which makes it a good candidate for transcription of the C9orf72 repeat expansion, which form various secondary structures on DNA and RNA level. This could also explain the consequential impact on the DPRs` levels. However, the impact on DPRs could also be on the account of SPFQ involvement in translational regulation, which was also previously shown.
Conclusions and scientific significance of the study: We identified multiple new interactors of less studied antisense RNA. Identified proteins are involved in multiple cell processes, and impairments in their function on account of their sequestration could be detrimental for neurons. We are first to identify the involvement of FARS and impairment of tRNA aminoacylation in C9orf72 mutation. Therefore, we significantly contributed to the understanding of the mechanisms impaired in the C9orf72 ALS and FTD. Furthermore, the involvement of tRNA aminoacylation in disease mechanisms opens new targets for therapeutic approaches. We are first to optimize the RNA-protein PLA method for detection of cytoplasmic interactions of antisense RNA. This is an important methodological contribution to the field of studying protein interactors of repeat expansion RNAs in cells, as these were so far limited to colocalizations of proteins with nuclear RNA foci. We also evaluated the impact of SFPQ expression levels on both sense and antisense RNA foci formation and on DPR synthesis. Therefore, we expanded the knowledge on the role of formerly known ALS and FTD protein SFPQ in C9orf72 mutation toxic mechanisms. The modulation of SFPQ expression level or its associated pathways represents a potential therapeutic approach in the C9orf72 ALS and FTD
Cloning and optimization of expression of recombinant proteins of Neat1 and G4C2 clusters for in vitro observation of their formation
No full textPrepisana heksanukleotidna razširitvena mutacija gena C9orf72 predstavlja osnovo za
izgradnjo toksičnih nemembranskih skupkov G4C2. Te se pogosto pojavljajo pri
družinskih različicah amiotrofične lateralne skleroze in frontotemporalne demence.
Mehanizmi nastanka toksičnih skupkov niso dobro poznani in bi lahko bili temelj novega
načina zdravljenja bolezni. Za raziskave skupkov so potrebni proteini, ki se kolokalizirajo
z mutiranim prepisom in tvorijo skupke G4C2. Zato smo v okviru diplomske naloge
pripravili plazmide za in vitro transkripcijo/translacijo in za izražanje proteinov SFPQ,
NONO, PABPC1, TDP-43 ter FUS v bakterijah E. coli BL21(DE3). Nemutiranim
različicam proteinov smo dodali fuzijski maltoza vezavni protein za povečano topnost
konstruktov in fluorescenčni protein NeonGreen. Plazmid je vseboval tudi
heksahistidinsko oznako in cepitveno mesto TEV za lažjo izolacijo konstruktov. Proteine
SFPQ, PABPC1, TDP-43 in FUS smo izražali pri 37 oC, 1 mM IPTG, tri ure in pri 30 oC,
0,5 mM IPTG, pet ur. Najboljši pogoji za izražanje proteina so bili pri nižji temperaturi
in nižji koncentraciji induktorja, ker se je takrat večina proteina nahajala v topni frakciji.
V prenizkih količinah za nadaljnjo izolacijo sta se izražala konstrukta SFPQ in SFPQNeonGreen. V nadaljevanju je potrebno preveriti izražanje konstruktov NONO, NONONeonGreen in NeonGreen-PABPC1 ter z uporabo Ni2+ ionov izolirati izražene proteine,
da dosežemo naš cilj.C9orf72 hexanucleotide repeat expansion transcript is the foundation on which toxic
membraneless G4C2 foci are built. These are common among patients with familial
amyotrophic lateral sclerosis and frontotemporal dementia. Mechanisms underlying the
formation of toxic G4C2 foci are unknown and could represent the basis for new
treatments. To research G4C2 foci proteins that localize and form these structures are
needed. For that reason, this work focuses on the production of plasmids for in vitro
transcription/translation and expression of SFPQ, NONO, PABPC1, TDP-43 and FUS
proteins in E. coli BL21(DE3) bacteria. A fluorescent NeonGreen protein was fused to
the wild type proteins along with maltose binding protein for increased construct
solubility. The plasmids also contained a hexahistidine tag and a TEV cleavage site to
assist with protein purification. SFPQ, PABPC1, TDP-43 and FUS were expressed at 37
oC with 1 mM IPTG for three hours and at 30 oC, with 0,5 mM IPTG for five hours.
Lower temperature expression yielded a better result since most of the protein was in the
soluble fraction of the cell lysate. Both SFPQ and SFPQ-NeonGreen were expressed in
too low a quantity for further protein isolation. Further work needs to check the expression
of NONO, NONO-NeonGreen, NeonGreen-PABPC1 and purify all of the constructs to
achieve our goal
Importance of phenylalanine for binding of TDP-43 protein to small RNAs
No full textTDP-43 je RNA- in DNA-vezavni protein, ki veže trans-aktivacijski odzivni element DNA. Ključen je pri regulaciji metabolizma RNA, prenosu mRNA, sintezi stresnih granul in pri drugih celičnih procesih. Običajno se TDP-43 nahaja v topni obliki, pri nekaterih nevrodegenerativnih boleznih, kot sta amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD), pa se kopiči v citoplazmi v obliki netopnih inkluzijskih telesc. Protein TDP-43, ki ga uvrščamo v družino heterogenih jedrnih ribonukleoproteinov (hnRNP), je sestavljen iz N-končne domene (NTD), dveh RNA-prepoznavnih motivov RRM1 in RRM2 ter iz neurejene C-končne domene (CTD). Vsebuje tudi jedrni lokalizacijski signal (NLS), signal za prenos iz jedra (NES) in več signalov za prenos v mitohondrije. Pri vezavi proteina TDP-43 na nukleinske kisline ima pomembno vlogo motiv RRM1, ki v zaporedju prepozna ponovitve UG oz. TG, motiv RRM2 pa poveča samo specifičnost vezave. V motivu RRM1 sta bistvena dva fenilalaninska aminokislinska ostanka, ki sodelujeta v π-π interakcijah nalaganja. V CTD se nahaja še osem fenilalaninov, ki mogoče tudi pomagajo pri stabilizaciji interakcije z RNA. V diplomskem delu smo v bakterijskem ekspresijskem sistemu T7 z avtoindukcijo izrazili protein TDP-43 in njegove mutante, kjer so bili v C-končni domeni in v motivih RRM1 ter RRM2 določeni fenilalaninski aminokislinski ostanki mutirani v levcinske. Proteine smo očistili z Ni-afinitetno kromatografijo. Izvedli smo tudi in vitro transkripcijo malih RNA, ki smo jih uporabili pri analizi vezave proteinov na RNA z metodo termoforeze na mikroskali (MST).TDP-43 is an RNA- and DNA-binding protein initially discovered for binding to the trans-active response element of DNA. It plays a key role in regulating RNA metabolism, mRNA transport, stress granule formation and other cellular processes. Under normal conditions, TDP-43 is found in a soluble form. However, in certain neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), it accumulates in the cytoplasm in the form of insoluble inclusion bodies. TDP-43 belongs to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and is composed of an N-terminal domain (NTD), two RNA recognition motifs (RRM1 and RRM2), and a disordered C-terminal domain (CTD). It also contains a nuclear localization signal (NLS), a nuclear export signal (NES) and several mitochondrial localization signals. In the binding of TDP-43 to nucleic acids, the RRM1 motif has a crucial role by recognizing UG or TG repeats in the sequence, while RRM2 enhances binding specificity. The RRM1 motif contains two essential phenylalanine residues that participate in π-π stacking interactions. There are also eight phenylalanines in the CTD, which may help stabilize the interaction with RNA. We successfully expressed TDP-43 and its mutants by auto-induction in a bacterial T7 expression system, in which some phenylalanine residues in C-terminal domain and in the RRM1 and RRM2 motifs of TDP-43 were mutated to leucine. The proteins were purified using Ni-affinity chromatography. We also performed in vitro transcription of small RNAs, which were used to analyze protein-RNA binding using the method of microscale thermophoresis (MST)
Deletion of SNORD116 gene cluster from the Prader-Willi locus in mammalian cell lines
Get PDFPrader-Willijev sindrom (PWS) je kompleksna multisistemska genetska bolezen, ki povzroča nevrološke, metabolne, endokrine in vedenjske motnje. Bolezen je posledica pomanjkanja izražanja očetovsko podedovanih genov na 15q11.2-q13 genomski regiji. Številne študije z mikrodelecijami so zožile kritično regijo PWS na gručo genov SNORD116, ki predstavlja glavno genetsko determinanto bolezni. Gensko gručo SNORD116 sestavlja 29 homologov, ki spadajo v družino malih nukleolarnih RNA s C/D ohranjenim motivom. Sekvenca SNORD116 ni komplementarna nobeni od kanoničnih RNA-tarč, zato ostaja biološka funkcija teh nekodirajočih RNA še neznana. Ravno iz tega razloga je ena izmed najpomembnejših nalog raziskav na tem področju iskanje in ovrednotenje nekanoničnih RNA-tarč iz družine SNORD116, ki bi bistveno pripomogle k določitvi vloge genske gruče pri patofiziologiji bolezni. V diplomskem delu smo s tehnologijo scCRISPR poskušali ustvariti celični liniji NTERA2/D1 in SH-SY5Y z izbitima genskima gručama SNORD116. Celična modela bi služila kot osnova za nadaljnje raziskovanje in validiranje najbolj verjetnih potencialnih tarč družine SNORD116. S pomočjo spletnih orodij smo na lokusu PWS izbrali tarčna zaporedja in načrtali sgRNA, ki smo jih nato s PCR amplificirali ter podaljšali, da smo dobili ustrezne komponente za sistem scCRISPR. Po tem, ko smo pripravili vse komponente in preverili njihovo ustreznost, smo z različnimi metodami optimizirali učinkovitost transfekcije pri obeh celičnih linijah. Plazmida in konstrukta sgRNA smo nato z načinom, ki se je izkazal za najbolj učinkovitega, vnesli v celice in preverjali, če so komponente sistema scCRISPR povzročile izbitje genske gruče SNORD116 v celičnem okolju. Za preverjanje smo uporabljali posamezne izolirane klone, pri katerih smo s specifičnimi začetnimi oligonukleotidi analizirali spremembe na genomski DNA na lokusu PWS. Izbitje genske gruče SNORD116 ni bilo uspešno pri nobeni od izbranih celičnih linij, najverjetnejši vzrok za negativen rezultat pa je prenizka učinkovitost transfekcije. Kljub temu, da nismo uspeli izdelati željenih celičnih modelov, smo v diplomskem delu uspešno vzpostavili sistem in metode potrebne za spreminjanje genoma s sistemom CRISPR/Cas9. Poleg tega smo optimizirali metode za preverjanje vnosa mutacij in selekcioniranja ter generiranja posameznih klonov. Naše ugotovitve bodo služile kot osnova za nadaljnje poskuse izbitja večjih genskih segmentov v celičnem okolju in s tem generiranjem modelov, ki nam bodo v prihodnje pomagali pri odkrivanju biološke vloge te enigmatične genske gruče.Prader-Willi syndrome (PWS) is a complex multisystemic genetic disorder with implications on the endocrine and neurologic systems, metabolism, and behaviour. PWS arises from the lack of expression of genes on the paternally derived chromosome 15q11.2-q13. Cases harbouring microdeletions narrowed the PWS critical region to the SNORD116 family, which is the primary genetic determinant of the PWS phenotype. SNORD116 gene cluster consists of 29 homologous genes that belong to the group of C/D-box small nucleolar RNAs. Since SNORD116 lacks obvious antisense elements against canonical RNA targets, its biological function remains largely unknown. For this reason, one of the most important tasks in this field of research is to identify the non-canonical RNA targets of SNORD116 that would significantly help determine the role of this gene cluster in the pathophysiology of the disease. Within this work, we wanted to create SNORD116 knock-out cell models using the scCRISPR method in NTERA2/D1 and SH-SY5Y cell lines. The cellular models would serve as a basis for further research and validation of the most probable potential targets of SNORD116. Firstly, we selected target sequences at the PWS locus and designed sgRNAs, which were then amplified and extended by PCR to obtain the appropriate components for the scCRISPR system. After we prepared and validated all components, we tried to optimize the transfection efficiency in both cell lines with various methods. Both plasmids and sgRNA constructs were then introduced into cells with the method that proved to be the most effective. To check if the scCRISPR system caused the desired deletion in target cells we analysed changes in genomic DNA at the PWS locus in individual isolated clones by PCR with specific primers. Unfortunately, the deletion of SNORD116 was unsuccessful in both of the selected cell lines, most likely due to low transfection efficiency. Despite the fact that we were not able to create the desired cell models, we successfully optimized the CRISPR/Cas9 system and methods needed for site-directed genome editing. In addition, we optimized methods to verify mutations and methods for the selection and generation of individual clones. We believe our work provides good basis for further knock-out experiments and thus generation of cell models that will help us discover the biological role of this enigmatic gene cluster
Structural characteristics of hexanucleotide repeat expansions from C9orf72 mutation
No full textAmiotrofična lateralna skleroza (ALS) je nevrodegenerativna bolezen, ki se kaže v odmiranju motoričnih nevronov v hrbtenjači, možganskem deblu in motoričnem korteksu. Posledica odmiranja nevronov je atrofija mišic in znaki odpovedi možganskih živcev. Vzroki za razvoj ALS še niso dobro pojasnjeni, vendar znanstveni dokazi kažejo, da imata genetika in okolje vlogo pri njenem razvoju. Na drugi strani nevropatološkega spektra je bolezen frontotemporalna demenca (FTD), ki prizadene frontalni in temporalni reženj možganov in se kaže v spremembah osebnosti in obnašanja. Tako kot pri ALS, tudi pri FTD natančen vzrok razvoja bolezni še ni pojasnjen. Glavna patologija, ki se pojavlja pri 90 % bolnikov z ALS in približno 60 % bolnikov s FTD, je agregacija jedrnega proteina TDP–43 v citoplazmi. V patologijo ALS so vključeni tudi drugi RNA–vezavni proteini. Glavni genetski vzrok je mutacija v genu C9orf72. Gre za heksanukleotidno razširitev (G4C2)n v prvem intronu gena C9orf72, ki se pri zdravih osebah ponovi do približno tridesetkrat, pri bolnikih pa je teh ponovitev nekaj deset do več tisoč. Predpostavljeni so trije mehanizmi vpliva mutacije na razvoj bolezni. Prvi je pomanjkanje proteina C9orf72, drugi je pridobljena toksičnost smernih (G4C2)n in protismernih (C4G2)n RNA, ki se prepišejo iz razširitve, ter tvorba RNA skupkov. Zadnji predpostavljeni mehanizem pa je pridobljena toksičnost dipeptidnih ponovitev, ki se iz smerne in protismerne RNA prepiše preko od ATG—neodvisne RAN translacije.
V sklopu diplomskega dela smo raziskovali strukture smerne in protismerne RNA ponovitve, ki so posledica mutacije v genu C9orf72. Podrobneje smo raziskali sekundarno strukturo ponovitev (G4C2)n in (C4G2)n, ki je odvisna od števila ponovitev. Rezultati ponujajo boljši vpogled v mehanizme pridobljene toksičnosti RNA. Z in vitro transkripcijo smo pripravili RNA, ki smo jo nato uporabili za strukturne študije. Z nuklearno magnetno resonanco (NMR) smo pokazali, da vzorca (G4C2)8 in (G4C2)48 tvorita G–kvadruplekse v prisotnosti K+ ionov.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in loss of motor neurons in the spinal cord, motor cortex and brainstem. As a result of neuronal loss muscle atrophy and signs of brain nerve weakness occur. The exact cause of ALS is yet to be determined, but scientific evidence has shown that genetics and environment have a role in degeneration of motor neurons and development of the disease. On the other side of the neuropathological spectrum is the disease frontotemporal dementia (FTD), which affects the frontal and temporal part of the brain and shows changes in behaviour and personality. Just like with the ALS, the cause of FTD is still unknown. In patients with ALS as wel as FTD, TDP–43 pathology was observed, where the RNA binding protein TDP–43 aggregates in the cytoplasm. Other RNA binding proteins are included in the development of ALS. The primary genetic cause for the diseases is the mutation in the C9orf72 gene, where a hexanucleotide repeat expansion GGGGCC appears in the first intron. Healthy individuals have around 30 repeats whereas individuals affected with the disease have ranging from ten to several thousand repeats. There are three proposed mechanisms of how the disease occurs. The first one is loss–of–function of the protein C9orf72, second is RNA toxicity, where the sense and antisense transcripts of the gene form RNA foci and last is DPR toxicity. Sense and antisense RNA can be translated through the process of RAN translation.
In this thesis, research was focused on structural determination of longer sense and antisense RNA repeat expansion, as a result of the mutation in C9orf72 gene. The thesis shows detailed research of the (G4C2)n and (C4G2)n secondary structures of different repeat lengths, which would give a better understanding how RNA toxicity mechanism works. Using in vitro transcription, we prepared RNA constructs for examination. We used NMR spectroscopy to show that r(G4C2)8 and r(G4C2)48 form G–quadruplexes in the presence of K+ ions
Identification of the stress granule formation mechanism induced in cells upon cold atmospheric plasma treatment
No full textHladna atmosferska plazma (HAP) je ioniziran plin s približno sobno temperaturo. HAP izkazuje različne fizikalno-kemične lastnosti, saj tekom pridobivanja HAP nastajajo s reaktivne spojine, nevtralni delci, elektromagnetno polje in UV sevanje. HAP je mogoče uporabiti v različne namene, posvetili pa smo se uporabi v medicini. Do sedaj je znano, da HAP povzroča nastanek stresnih granul (SG) po od eIF2α-odvisni poti, vendar točen mehanizem aktivacije integriranega celične odziva (ISR) s strani HAP še ni poznan. Glede na dosedanje poznavanje delovanja HAP vemo, da se ob delovanju HAP tvorijo kisikove in dušikove reaktivne spojine. Z dodatkom znanega antioksidanta kverceta k celični liniji SH-SY5Y Flp-In mScarletI-G3BP1-Myc, smo preverili vpliv oksidativnega stresa na tvorbo SG ob delovanju HAP. Pri tem smo predvidevali, da kvercetin zavira aktivacijo kinaze HRI, ki je odgovorna za nastanek SG ob prisotnosti oksidativnega stresa. Nastanek in lastnosti SG smo opazovali v celični liniji SH-SY5Y Flp-In mScarletI-G3BP1-Myc, ki ima na protin G3BP1 vezan rdeči fluorescenčni protein mScarletI. Protein G3BP1 je eden izmed proteinov v SG, zato se ob delovanju stresa rekrutira v SG, kar opazujemo s fluorescenčno konfokalnim mikroskopom. HAP smo pridobivali s plazemsko šobo pri konstantnem dotoku argona. Pokazali smo, da z dodatek kvercetina k celicam zmanjša delež celic s stresnimi granulami, hkrati pa vpliva na stresni stimulus HAP. Tako smo potrdili vpletenost kisikovih in dušikovih reaktivnih spojin v tvorbi SG. Vpliv kvercetina na fosforilacijo eIF2α nismo dokazali.Cold atmospheric plasma (CAP) is a near-room temperature ionized gas that is widely used in biomedicine. CAP cause physicochemical effects, as various processes take place during the generation of CAP, such as reactive compounds, neutral particles, electromagnetic fields and UV radiation. It is well established that CAP causes the formation of stress granules via the eIF2α-phosphorylation dependent pathway. However, it is unknown which of the integrated stress response related kinases is responsible for the phosphorylation of eIF2α. We can assume that CAP is responsible for oxidative stress due to the generation of reactive oxygen and nitrogen species by the application of CAP. Quercetin is a known antioxidant, so we assumed that quercetin may inhibit the activation of heme-regulated inhibitor kinase, which is activated during oxidative stress. We used the cell line SH-SY5Y Flp-In mScarletI-G3BP1-Myc, in which the fluorescent protein mScarletI is fused to one of the core proteins of stress granules, G3BP1. The source of CAP was a plasma jet with constant argon flow. We demonstrated that quercetin significantly reduced the number of cells with stress granules when incubated for one hour before treatment with CAP. Quercetin also acts on strength of stress stimuli caused by CAP and on affects stress granule morphology. We did not detect any effect of quercetin on eIF2α phosphorylation
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