In the last decade, there has been increasing research into the function and the mechanism of pathogenicity of membraneless organelles (MLOs). MLOs are essential for RNA metabolism including RNA transportation, transcription, translation, and degradation. Ribonucleoproteins (RNPs) participate in these processes and are key components of MLOs. For example, the RNPs hnRNPA2 and hnRNPF bind myelin basic protein mRNA, and this complex associates with a type of MLO called an RNA transport granule, which facilitates transportation to oligodendrocytes for translation. Similarly, the RNPs hnRNPU and LSM14 bind and localize RNA, and this complex associates with a type of MLO called a processing body, which facilitates RNA destabilization and decay. Another example is that the RNPs FUS and TDP-43 bind mRNA, and this complex associates with at type of MLO called a stress granule which forms and disappears in response to stress stimuli. Increasing studies are performed to span our knowledge of the relationship between MLOs and neurodegenerative diseases. RNPs often contain two types of functional domains: an RNA binding domain (RBD) for recognizing RNA and a low complexity domain (LCD) to facilitate MLO formation; most attention in neurodegeneration research has focused on the LCD. LCDs are composed of a limited repertoire of amino acid types such Glycine, Tyrosine, and polar residues. LCDs of many RNPs, including FUS and LSM4, form a separate, dense phase in vitro similar to cellular MLOs and can further self-assemble into stable amyloid-like fibrils with cross-β structures. Their propensity to phase separate implicates the LCDs of RNPs as being principle drivers of the formation and dynamics of MLOs. Mutations in the LCDs can accelerate their self-assembly rates, leading to hyper-stable MLOs which contribute to diseases. Over 50 mutations are found to be located in the LCDs and convert functional amyloid-like fibrils into pathogenic amyloid, but only a few atomic structures of the LCDs are known due to their lack of tertiary structures. Knowing the structures of the LCDs can help us better understand their functions and pathogenic mechanisms. So far, over 40 amyloid fibril structures are determined to atomic resolutions, but none before the work described here gives a clear structural explanation of its pathogenic mechanism.
To fill this gap, we used cryoEM and determined the atomic structures of both wildtype and the variant hnRNPA2 LCD, an RNP component of RNA transport granules and stress granules. The wildtype structure of hnRNPA2 LCD shares intrinsic structural and energetic properties with other RNPs such as FUS: it has only one fibril morphology with a single protofilament and it is stabilized by hydrogen bonding and polar interactions. Wildtype hnRNPA2 has a poor solvation energy value, caused by the scarcity of residues that adopt the β-sheet conformation in the structure, which thereby weakens fibril stability. The variant structure turns out to be very different from wildtype in three ways: 1) It has at least 6 morphologies with two or more protofilaments. 2) It has a solvation energy value close to other pathogenic amyloids such as Tau. 3) More residues adopt β-sheet conformations. Compellingly, the variant structures of hnRNPA2 LCD offer evidence for its pathogenic mechanism. A loss of function arises because the variant’s PY nuclear localization signal (PY-NLS) is hidden in the fibril core, disrupting its ability to bind to transporters and be localized into the nucleus. Over half of the inward facing residues in the wildtype structure turn outward facing in the variant structure, exposing new surface epitopes that could potentially contribute to the variant’s toxicity. Thermostability and phase separation assays also confirm our structural results that the variant hnRNPA2 LCD fibrils are more stable than wildtype fibrils. We believe that the variant hnRNPA2 LCD fibril structure is unusual among other pathogenic amyloid fibrils because it suggests an obvious mechanism for pathogenicity and may shed light on analogous mutational conversions occurring in other neurological diseases.
