Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration

Abstract

mRNA transport in neurons is a ubiquitous process but has been often overlooked as a contributor to disease. Mutations of transport granule protein hnRNPA2 cause hereditary proteinopathy of neurons, myocytes, and bone. Here, we examine transport granule component specificity, assembly/disassembly, and the link to neurodegeneration. hnRNPA2 transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet they each partition specifically into hnRNPA2 liquid phases. hnRNPA2 tyrosine phosphorylation dissociates granule interactions by reducing hnRNPA2 phase separation and preventing partitioning of hnRNPF and ch-TOG; tyrosine phosphorylation also decreases aggregation of hnRNPA2 disease mutants. A C. elegans model of hnRNPA2 D290V-associated neurodegeneration exhibits TDP-43 ortholog-dependent glutamatergic neurodegeneration.Expression of the tyrosine kinase that phosphorylates hnRNPA2 reduces glutamatergic neurodegeneration. The evidence for specific partitioning of granule components as well as disruption of these interactions and reduction of neurodegeneration by tyrosine phosphorylation suggest transport granule biology has a role in the pathogenesis of neurodegeneration.

Weak binding to the A2RE RNA rigidifies hnRNPA2 RRMs and reduces liquid–liquid phase separation and aggregation

“…Indeed, while both D290V and P298L formed aggregates over time, the addition of RNA prevented formation of both liquid droplets and solid aggregates at these conditions (Figure 4B , C). This prevention of aggregation was even greater than that exerted by partial tyrosine phosphorylation ( 33 ), as here neither D290V nor P298L showed any droplets in the presence of RNA.…”

“…the LLPS assay is performed at concentrations below the dissociation constant), a majority of the RNA is not predicted to bind two copies of the protein simultaneously, perhaps contributing to the lack of stimulation of LLPS by addition of rA2RE21. Here, we performed all LLPS assays at 15 μM hnRNPA2 FL as hnRNPA2 FL undergoes robust LLPS at that concentration ( 10 , 33 ). Despite the fact that at these concentrations (given the binding affinities we found for rA2RE11 and rA2RE21) only a fraction of hnRNPA2 RRMs would be expected to bind RNA, we do see a profound disruption of LLPS by addition of RNA on LLPS.…”

“…In contrast to previous reports ( 5 ), we find that both the 11mer and 21mer RNA oligomers bind to hnRNPA2 1–189 weakly (Figure 5 ), with a binding affinity on the order of tens of micromolar. Given that we have recently shown hnRNPA2 is highly prone to phase separate and aggregate and requires careful use of solubility tags and RNA removal to prevent pre-aggregation ( 10 , 22 , 30 , 33 ), it is possible that self-interactions between full-length hnRNPA2 molecules (e.g. pre-formed aggregates) and protein-surface interactions complicated previous bio-sensor measurements performed with full-length recombinant protein.…”

“…The impact of physiological arginine methylation, which alters hnRNPA2 LC self-interaction and LLPS ( 10 ), on hnRNPA2 LC interactions with RNA will be important to study in the future. Though our focus here is on the RNA-binding properties of the hnRNPA2 RRMs to the A2RE, it is possible that the in vitro LLPS experiments may not represent the effect of RNA on hnRNPA2 phase separation in cells where other protein and RNA interactions may cooperate with the A2RE interaction with hnRNPA2 RRMs to stabilize a granule ( 33 ).…”

A predictive coarse-grained model for position-specific effects of post-translational modifications on disordered protein phase separation

“…Therefore, computational studies can be an effective approach to screen a large number of PTM patterns and predict their role on regulating the molecular properties and phase separation of proteins (30,31). All-atom explicit solvent simulations readily incorporate posttranslationally modified amino acids (32)(33)(34)(35)(36) and have been used to probe the contacts leading to phase separation (37,38). However, the ability of atomistic force-fields to accurately capture the properties of IDRs depends strongly on parameterization (39,40).…”