Periodic Reporting for period 2 - RNPdynamics (Multivalent interactions driving RNP dynamics in development and disease)
Okres sprawozdawczy: 2021-07-01 do 2022-12-31
Mutations in many RNA-binding proteins cause neurodegenerative diseases, and most of these mutations locate to disordered regions and can change condensation properties of these proteins. Condensation has so far been monitored mainly in vitro (such as formation of liquid droplets) or by imaging (such as formation of RNA granules in cells). However, little is known about the impact of such changes on the RNA binding functions of these proteins.
Understanding the way how disordered regions contribute to RNA binding is essential in order to understand how mutations in these regions act, and how they initiate the early stages of molecular changes that eventually lead to neurodegeneration. To address this question, we proceeded along five objectives:
1. identifying the functions of disorder-containing RNA-binding proteins during stem cell differentiation.
2. Assessing the importance of multivalency of the bound RNA regions for the formation of protein-RNA condensates.
3. Manipulating protein-RNA assembly through multivalent RNA regions.
4. Manipulating the protein disorder-dependent assembly and dynamics of protein-RNA complexes.
5. Assessing the species-specific roles of multivalent protein-RNA complexes.
Understanding the way how disordered regions contribute to RNA binding is essential in order to understand how mutations in these regions act, and how they initiate the early stages of molecular changes that eventually lead to neurodegeneration. To address this question, we proceeded along five objectives:
1. identifying the functions of disorder-containing RNA-binding proteins during stem cell differentiation.
2. Assessing the importance of multivalency of the bound RNA regions for the formation of protein-RNA condensates.
3. Manipulating protein-RNA assembly through multivalent RNA regions.
4. Manipulating the protein disorder-dependent assembly and dynamics of protein-RNA complexes.
5. Assessing the species-specific roles of multivalent protein-RNA complexes.
We have achieved the planned milestones on all five work packages, some of them through collaborations with other teams. We developed an improved iCLIP prototol (iiCLIP) that can be performed non-radioactively and yields highly reproducible and quantitative data (Lee et al, bioRxiv, 2021). To be able to exploit such data for comparative analyses of mutant variants of TDP-43, we have developed new computational method for identification and analysis of enriched binding motifs called PEKA (Kuret et al, bioRxiv, 2021) and a method for assignment of binding regions based on such motifs. We used iiCLIP to characterize a highly multivalent RNA binding region of TDP-43 in an intron of a gene called UNC13A that contains disease-associated variants and a cryptic exon repressed by TDP-43 (Brown et al, Nature, 2022). Moreover, we used ribosome profiling to characterise the mechanism whereby a natural antisense transcript (NAT) affects translation of the MAPT mRNA (Simone et al, Nature, 2021).
Most importantly, we addressed the long-standing question of whether TDP-43 condensation is required for its RNA binding specificity and function, and how this relies on multivalent features of bound RNA regions (Hallegger et al, Cell, 2021). We created variants of TDP-43 containing mutations in its disordered regions with a gradient of condensation properties as evident by in vitro phase separation, and by imaging the condensates of TDP-43 in cells. We then used comparative iCLIP to find that the condensation of TDP-43 is required for its efficient binding to specific, long RNA regions that are highly multivalent with dispersed binding motifs. Thereby, we could explain how the condensation propensity of TDP-43 affects regulation of a select subset of 3’UTR isoforms, including autoregulation of TDP-43 itself.
Most importantly, we addressed the long-standing question of whether TDP-43 condensation is required for its RNA binding specificity and function, and how this relies on multivalent features of bound RNA regions (Hallegger et al, Cell, 2021). We created variants of TDP-43 containing mutations in its disordered regions with a gradient of condensation properties as evident by in vitro phase separation, and by imaging the condensates of TDP-43 in cells. We then used comparative iCLIP to find that the condensation of TDP-43 is required for its efficient binding to specific, long RNA regions that are highly multivalent with dispersed binding motifs. Thereby, we could explain how the condensation propensity of TDP-43 affects regulation of a select subset of 3’UTR isoforms, including autoregulation of TDP-43 itself.
We will continue developing new advanced experimental (iiCLIP) and computational methods in a manner that is mutually reciprocal, thus leading to hypotheses that we test with further biochemistry, molecular and cellular biology. This broad combination of techniques enables us to approach important questions in new and fundamental ways. Primarily, we will continue studying how changes in the condensation propensity of RNA-binding proteins affect a selective subset of RNAs bound by this protein, and use this to understand how mutations in RNA-binding proteins act, and how they could either contribute to the early stages of molecular changes that eventually lead to neurodegeneration or to species-specific functions. We will also develop new potential therapeutic approaches to modulate condensation propensity of protein-RNA complexes and thereby selectively control RNA regulation.