Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation
Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets
Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets
View: Text | PDF
Research Article Genetics Neuroscience

Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation

Abstract

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.

Authors

Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets

×

Figure 4

hnRNPA1 variants can exhibit altered propensity for fibrillization and LLPS.

Options: View larger image (or click on image) Download as PowerPoint
hnRNPA1 variants can exhibit altered propensity for fibrillization and L...
(A) ZipperDB calculates the propensity of hexapeptide fragments to form steric zippers (48). Steric zippers, which are self-complementary β-sheets that form the backbone of amyloid fibrils, are predicted to form when the Rosetta energy of a hexapeptide is below the empirically determined “high fibrillization propensity” threshold of −23 kcal/mol (48). P288A introduces a potent steric zipper (285-SSGAYG-290) that could increase the fibrillization propensity. G304Nfs*3 deletes several potent steric zippers, which could reduce fibrillization propensity. (B and C) GST-TEV-hnRNPA1 and disease variants (5 μM) were incubated with TEV protease in A1 assembly buffer to initiate fibrillization. Reactions were agitated at 1200 rpm or 0–24 hours at 25°C. Fibrillization was monitored by electron microscopy (B). Scale bar, 0.5 μm. Alternatively, hnRNPA1 fibrillization kinetics were determined by sedimentation analysis (C) where the amount of hnRNPA1 in the pellet fraction was quantified. Values represent average ± SEM (n = 3–6). (D) Representative differential interference contrast (DIC) microscopy images of hnRNPA1 droplets formed by different hnRNPA1 variants. Droplets were formed by combining the indicated hnRNPA1 variant at the indicated concentration in a LLPS buffer and were imaged immediately after all components had been added. Scale bar: 25 μm. (E) Phase diagram of hnRNPA1 variants showing the hnRNPA1 concentrations where LLPS occurs. hnRNPA1 and hnRNPA1P288A form droplets at concentrations of 2.5 μM or higher. hnRNPA1*321Eext*6 forms droplets at concentrations of 5 μM or higher. hnRNPA1G304Nfs*3 forms droplets at concentrations of 20 μM or higher. At each concentration, a colored circle indicates droplet formation, whereas a black asterisk indicates no droplets.