Departments

Šifra projekta / Project code: J4-50145

Vodja: dr. Miha Modic

1. Vsebinski opis projekta:

   Brezmembranski kompartmenti, ki se tvorijo ob kondenzatih lncRNA, so ključnega pomena za razvoj in so tesno povezani z nevrodegenerativnimi boleznimi. Potrebno jih je razmejiti in modulirati njihovo sestavo, da bi razumeli delovanje teh oddelkov. V predlaganem projektu bomo raziskovali, kako lahko ena specifična lncRNA, NEAT1, deluje kot "seme" za sestavljanje kompleksa ribonukleoproteinov z visoko lokalno kondenzacijo v brezmembranski kompartment imenovan parapega. NEAT1 spodbuja fazno ločevanje RNA vezavnih proteinov, kar je močno odvisno od raznolikosti interakcij, ki jih tvorijo intrinzično neurejena območja (ang. intrinsically disordered regions, IDR) RNA vezavnih proteinov (ang. RNA Binding Proteins, RBP), ki so sestavni del parapeg. Ti IDR-ji parapeg RBP so pogosto mesta mutacij, ki povzročajo bolezni, mutirani RBP pa imajo tudi povečano nagnjenost k prehodu iz tekoče v trdno fazo, kar je pogost znak več nevrodegenerativnih bolezni. Predvidevamo, da post-translacijske modifikacije IDR-jev narekujejo potencial tvorjenja kompartmentov ob kondenzatih lncRNA in so povezani z večanjem sposobnosti RBP-jev, da imajo na različnih stopnjah razvoja več vlog v regulacijskih omrežjih RNA. Obravnavali bomo dve osnovni hipotezi:
   1. Sestavljanje endogenih membranskih kompartmentov je regulirano tako, da je tesno povezano s prehodi celice v različne stopnje razvoja, in
   2. Spremembe v intrinzično neurejenih regijah vplivajo na delovanje interagirajočih RBP-jev in s tem uravnavajo sklop brezmembranske kompartmente, ki lahko prispevajo k prehodom celice v različne stopnje razvoja.

   Some long noncoding RNAs (lncRNAs) have a capacity to scaffold high concentration of RNA-binding proteins (RBPs) and assemble into membraneless compartments. These are characterised by a tendency for phase separation when reconstituted in vitro, they are visible as granules under light microscope and act as liquid droplets with high mobility when studied in cells. Membraneless compartments are essential for development and play a key role in human diseases associated with liquid-liquid phase separation (LLPS). It is imperative to delineate and modulate their composition in order to understand the function of these compartments. Here, we will ask how one hallmark lncRNA scaffold, NEAT1, may act as a “seed” to assemble a ribonucleoprotein complex (RNP) with high local condensation into a membraneless compartment called paraspeckles and how this impacts the differentiation propensity of human embryonic stem cells (ESCs). It namely remains unknown how any of the lncRNA-scaffolded biomolecular compartments steer cell fate decisions, which opens the next frontier. NEAT1 promotes a phase separation of RBPs, which heavily depends on the valency of interactions formed by the intrinsically disordered regions (IDRs) of RBPs. These IDRs of paraspeckle RBPs are hotspots for neurodegeneration-causing mutations and posttranslational modifications (PTMs). We propose that PTMs of IDRs dictate the capacity of RBPs to assume multiple roles in RNA regulatory networks at different developmental stages. In this proposal, we extend the toolkit to investigate how can programming of lncRNA-dependent remodelling of RNPs drive cell-fate transitions by integrating cell engineering, iCLIP, transcriptomics, proteomics, and NMR spectroscopy along with our newly developed proximity labelling method. Our approach towards elucidating mutations in IDRs that affect cell fate transitions by altering the recruitment of the corresponding RBPs to paraspecies is a bottom-up approach: we start by observing changes in composition and PTM-dependent protein interactions and then use a computational approach to infer common principles behind these changes. This narrows down the regulatory mechanism hypotheses that will be the subject of more detailed structural studies and further investigation by programming synthetic nuclear condensates in the ESC model of differentiation. We will explore two primary hypotheses:
   1. The assembly of membraneless compartments is regulated, so that it is tightly coupled to cell fate transitions in human development, and
   2. Modifications in IDRs affect the function of interacting RBPs, thereby regulating membraneless compartment assembly that can contribute to cell fate transitions.

   
   Sodelujoče organizacije:
   
   Institut Jožef Stefan
   
   
   Osnovni podatki glede financiranja:
   Projekt financira ARIS v okviru cenovne kategorije D za obdobje 3 let v obsegu 1960 letnimi urami. Pričetek financiranja je 1. 10. 2023.
   The project is co-financed by ARIS with 1960 annual hours of price class D for a period of 3 years. Funding starts on 1st of October 2023.
   
   Sestava projektne skupine s povezavami na SICRIS
   Na Kemijskem inštitutu v projektni skupini sodelujejo / At the National Institute of Chemistry the project group includes:

   Miha Modic; SICRIS 50784;  cris.cobiss.net/ecris/si/sl/researcher/46965
   Anja Trupej; SICRIS 57147; cris.cobiss.net/ecris/si/sl/researcher/54119
   Jernej Ule; SICRIS 34667; cris.cobiss.net/ecris/si/sl/researcher/39367
   Maja Marušič; SICRIS 34525; cris.cobiss.net/ecris/si/sl/researcher/39140
   Janez Plavec; SICRIS 10082; cris.cobiss.net/ecris/si/sl/researcher/6890

2. Faze projekta in njihova realizacija

   
   Cilj 1. Kako parapege vplivajo na sestavljanje jedrnih RNP za spodbujanje izstopa iz pluripotentnosti?
   Cilj 2. Kako parapege vplivajo na delovanje interaktivnih RBP-jev?
   Cilj 3. Kako spremembe IDR vplivajo na sestavljanje in delovanje parapeg?

   
   Aim 1. How do endogenous and synthetic paraspeckles affect the assembly of nuclear RNPs to promote exit of pluripotency?
   Aim 2. How do paraspeckles affect the function of interacting RBPs?
   Aim 3. How do modifications in IDRs affect the assembly and function of paraspeckles?

3. Bibliografske reference, ki izhajajo neposredno iz izvajanja projekta
   
   Bo dopolnjeno tekom projekta/to be added