Mechanistic View of hnRNPA2 Low-Complexity Domain Structure, Interactions, and Phase Separation Altered by Mutation and Arginine Methylation

Abstract

hnRNPA2, a component of RNA-processing membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons (bearing features of amyotrophic lateral sclerosis [ALS] and frontotemporal dementia), muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction and the distinct effects of small chemical changes-disease mutations and arginine methylation-on these assemblies. The hnRNPA2 low-complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Co-aggregating in disease inclusions, hnRNPA2 LC directly interacts with and induces phase separation of TDP-43. Conversely, arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations.

TDP-43 α-helical structure tunes liquid-liquid phase separation and function

“…At low CTD concentrations (50 µM and below) the apparent diffusion coefficient for WT CTD remains largely unchanged (8.8± 0.1 · 10 -11 m 2 s -1 ) ( Figure 3C), suggesting that this value reflects the diffusion coefficient of monomeric CTD. This diffusion coefficient corresponds to an approximate radius of gyration of 2.8 nm in dilute aqueous solution, close to that of the similar-length low complexity (LC) domain of hnRNPA2 43 and significantly more extended than the globular reference protein lysozyme (2.03 nm) ( Figure S3A), which is of almost identical molecular weight to TDP-43 CTD. At concentrations above 50 μM, however, the apparent diffusion coefficient of the CTD sample decreases, consistent with CTD assembly into higher order species.…”

“…Similarly, the contacts formed by TDP-43 CTD conserved region are also heterogeneous 32 , unlike amphiphilic α-helices that form ordered, rigid bundles. Indeed, CTD assembly may underlie the ability of this region of TDP-43 to interact with splicing factor partners including hnRNPA231,43 . Hence TDP-43 CTD conserved region serves as a dynamic and heterogeneous binding module to enhance TDP-43 phase separation.The identification of single, specific sites that can be used to substantially alter the assembly and material properties of cellular droplets offers an important opportunity to use TDP-43 CTD as a module to tune phase separation in engineered fusion proteins.…”

Protein Phase Separation: A New Phase in Cell Biology

“…Work from different labs has shown that both serine and tyrosine phosphorylation can control phase separation [12,52,89,90], and the same holds true for arginine methylation [49,91] and sumoylation [80]. Importantly, the activity of the dual specificity kinase DYRK3, which partitions into SG, was shown to be necessary for SG dissolution [92], suggesting that there might be specific cellular switches controlling these processes.…”

m6A facilitates YTHDF‐independent phase separation

“…2 This phenomenon is similar to the mixing of water and oil or can be imagined as the process of raindrop formation over an umbrella on rainy days. 5 Recently, Ries et al 6 To address whether m 6 A regulates the phase separation of DF2 protein, the authors found that in m 6 A methyltransferase 14-knockout mouse embryonic stem cells, the formation of endogenous stress granules took place normally after exposure to heat shock; however, the localization of DF2 proteins in stress granules was significantly reduced. 3 Cells are separated from each other by phase separation to achieve a variety of biological functions.…”

“…4 The multivalent interaction of intracellular biological macromolecules is common and affected by some modifications, such as phosphorylation and methylation. 5 Recently, Ries et al 6 To address whether m 6 A regulates the phase separation of DF2 protein, the authors found that in m 6 A methyltransferase 14-knockout mouse embryonic stem cells, the formation of endogenous stress granules took place normally after exposure to heat shock; however, the localization of DF2 proteins in stress granules was significantly reduced. The localization of DF2 to stress granules under cellular stress is dependent on its modification with m 6 A.…”