Opredelitev vloge protismernega zapisa RNA gena C9orf72 mutiranega pri amiotrofični lateralni sklerozi in frontotemporalni demenci

Malnar, Mirjana (author), Rogelj, Boris (mentor)

Uvod: 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.

• Language: Slovenian
• Keywords: ALS, FTD, protismerna RNA, FARSA, aminoacilacija tRNA, SFPQ, DPR
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2021
• PID: 20.500.12556/RUL-127270
• COBISS.SI-ID: 137789443
• Publication date in RUL: 01.06.2021

Secondary language

• Language: English
• Title: Role of antisense RNA transcript from C9orf72 mutation associated with amyotrophic lateral sclerosis and frontotemporal dementia

Abstract

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 repeats; however, 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.

• Keywords: ALS, FTD, antisense RNA, FARSA, tRNA aminoacylation, SFPQ, DPR