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Micellar TIA1 with Folded RNA Binding Domains As a Model for Reversible Stress Granule Formation

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Micellar TIA1 with Folded RNA Binding Domains As a Model for Reversible Stress Granule Formation Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation Keith J. Fritzschinga, Yizhuo Yanga, Emily M. Poguea, Joseph B. Raymanb,c, Eric R. Kandelb,c,d,e,f, and Ann E. McDermotta,1 aDepartment of Chemistry, Columbia University, New York, NY 10027; bDepartment of Neuroscience, College of Physicians and Surgeons, Columbia University, New York, NY 10032; cDepartment of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, NY 10032; dHHMI, Columbia University, New York, NY 10032; eZuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10032; and fKavli Institute for Brain Science, Columbia University, New York, NY 10032 Contributed by Ann E. McDermott, September 24, 2020 (sent for review April 20, 2020; reviewed by Bernd Reif and Robert Tycko) TIA1, a protein critical for eukaryotic stress response and stress bound to the dinucleotide UU-RNA (PDB ID code 5O3J), and granule formation, is structurally characterized in full-length form. TIA1-RRM2,3 (PDB ID code 2MJN). RRM1 appears to con- TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal tribute little to RNA binding, which may be explained by the low-complexity domain, sometimes referred to as a “prion-related negatively charged residues in the RNP1 motif within RRM1. A domain” or associated with amyloid formation. Under mild condi- model of rigid RRM domains with flexible linkers was used to tions, full-length (fl) mouse TIA1 spontaneously oligomerizes to interpret scattering data and show that RRM2 and RRM3 as- form a metastable colloid-like suspension. RRM2 and RRM3, sociate more closely with each other and more so after RNA known to be critical for function, are folded similarly in excised binding than with RRM1. However, the effects of the LCD on domains and this oligomeric form of apo fl TIA1, based on NMR the fl structure have remained elusive due to experimental chemical shifts. By contrast, the termini were not detected by NMR challenges. and are unlikely to be amyloid-like. We were able to assign the It has been reported that the LCD of TIA1 can cause phase NMR shifts with the aid of previously assigned solution-state shifts separation (14). In vivo, phase separation of groups of func- for the RRM2,3 isolated domains and homology modeling. We tionally related, locally concentrated proteins and nucleic acids present a micellar model of fl TIA1 wherein RRM2 and RRM3 are (14) is believed to lead to the formation of membraneless or- colocalized, ordered, hydrated, and available for nucleotide bind- ganelles (biomolecular condensates), such as stress granules BIOPHYSICS AND COMPUTATIONAL BIOLOGY ing. At the same time, the termini are disordered and phase sep- (15). Many cellular condensates contain proteins with RNA- arated, reminiscent of stress granule substructure or nanoscale binding domains, including Cajal bodies, P bodies, and SGs liquid droplets. (15). Misregulation of phase separation has been implicated in several human disease-related functions (8, 9). solid-state NMR | stress granule | RNA binding protein | membranelles Many proteins with LCDs also spontaneously partition into organelles | prion separate phases or form gels at high concentrations in vitro, potentially providing a model for in vivo phase separation. The cell intracellular antigen-1 (TIA1) has multiple roles within in vitro systems share important properties with the corre- Tcells, including a critical role in stress granule (SG) formation sponding in vivo systems. Both can undergo transitions and dis- during eukaryotic cellular stress response (1–3) and translation play a continuum of mechanical properties from liquid-like regulation (4–6). SGs appear in cells exposed to stressors, such as droplets to glassy (16), solid-like particles. Liquid–liquid droplets pH, oxidation, and temperature changes, and contain stalled are typically micrometer, morphologically spherical domains that preinitiation RNA–protein complexes. They have been hypoth- exhibit liquid-like dynamics in their rapid recovery from photo- esized to act as a decision point in mRNA processing by helping bleaching and solution-state NMR spectra (17). Many liquid to guide homeostasis-restoring protein expression or begin ap- droplets or condensates in vitro are metastable and transform optosis. Although sometimes associated with misfolded protein over time (16, 18) in a process referred to as hardening or aggregates, SG components dissolve and regain function more quickly than other aggregates after the stress is removed (7). Significance TIA1 has three RNA recognition motifs (RRM1, RRM2, RRM3) known to bind RNA with relatively little sequence Structural characterization of fl mouse TIA1 (43 kDa monomer) specificity. TIA1 has a C-terminal low-complexity domain (LCD) with solid-state NMR reveals structured functional domains enriched with asparagine and glutamine that has been referred and a disordered low-complexity domain. These data are con- to in the literature as a prion-related domain (PRD) because of sistent with a micellar form of apo fl TIA, unexpected based on its sequence similarity to amyloid- or prion-forming proteins. much previous literature. We expect that these models of TIA1 Proteins associated with SGs have also been linked to several and the NMR approaches will help us understand many similar human diseases, some characterized as protein misfolding dis- proteins and their roles in the cellular stress response. orders. A mutation within the LCD of TIA1 is the diagnostic marker for Welander distal myopathy (8, 9), and several other Author contributions: K.J.F. and A.E.M. designed research; K.J.F., Y.Y., and E.M.P. per- formed research; J.B.R. contributed new reagents/analytic tools; K.J.F., Y.Y., and A.E.M. TIA1 mutations are linked to amyotrophic lateral sclerosis (10). analyzed data; K.J.F., E.M.P., J.B.R., E.R.K., and A.E.M. wrote the paper; and E.R.K. and Despite its importance, little is known about the full-length (fl) A.E.M. supervised. or oligomeric form(s) of TIA1 or about the LCD. The RRM Reviewers: B.R., Technical University Munich; and R.T., National Institute of Diabetes and domains have been structurally characterized (11–13), giving Digestive and Kidney Diseases. insight into structure, dynamics, binding, and function. Several The authors declare no competing interest. excised TIA1-RRM domain constructs were characterized with Published under the PNAS license. small-angle scattering and liquid-state NMR; the LCD was ex- 1To whom correspondence may be addressed. Email: [email protected]. cluded from the constructs used in prior published structural This article contains supporting information online at https://www.pnas.org/lookup/suppl/ studies (11, 13). NMR was used to solve the structure of TIA1- doi:10.1073/pnas.2007423117/-/DCSupplemental. RRM1 (Protein Data Bank [PDB] ID code 5O2V), TIA1-RRM2 www.pnas.org/cgi/doi/10.1073/pnas.2007423117 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 maturation (7, 10, 16, 19). Analogously, membraneless organ- scattered light and was denser than the surrounding solution, as elles can undergo transitions in vivo during regulated maturation shown in Fig. 1C. The separation (monitored by scattering) was processes (15, 20, 21). Thus, it has been suggested that mis- accelerated by stirring or agitation. The protein-enriched phase folded, amyloid-like fibrils and aggregates form during matura- could be sedimented with excellent yield using gentle centrifu- tion of the membraneless organelles (7), suggesting a role for gation (gravity overnight with no agitation or with moderate condensates in templating the formation of disease-related fibrils centrifugation as described in Methods); no residual soluble (22). Furthermore, the multiphase in vitro suspensions can be TIA1 in the supernatant was detected by ultraviolet-visible compared to colloids, in that they are homogeneously distributed, stable multiphase suspensions whose formation is controlled by spectroscopy. The phase separation is reversible; dilution and salt concentration, viscogens, and temperature. However useful gentle agitation or an increase in ionic strength rehomogenize these analogies are, it is important to note that the in vitro systems the solution. The fact that the protein sediments at low g force are, of course, highly simplified compared to the in vivo situation. suggests that under these mild aqueous conditions, the fl protein The in vivo systems are subject to important biological control forms a high-order oligomer. The dense protein phase following over their formation and dissolution and include many other centrifugation is reminiscent of a metastable colloid-like sus- components such as nucleic acids and other proteins. pension. It forms reversibly in a manner dependent on cosolutes, The LCD/PRD of TIA1 has primary sequence similarity to the appears to have dispersed particles, and is stable for many hours. better-characterized SUP35 prion protein and is also similar to We think of the TIA1 samples prepared in this way as colloidal – other amyloid-forming proteins (23 25). Atomic force and homo-oligomeric apo (absence of RNA) wild-type full-length electron microscopy (EM) have been used to show that TIA1 mouse TIA1 and refer to this kind of preparation whenever dis- forms fibers under some conditions (26, 27). Congo red and cussing fl TIA1 (unless otherwise specified). While the properties thioflavin T binding assays have been used to suggest TIA1 forms a cross-β amyloid (26, 28). Sup35 and FUS are proteins with a in many ways resembled colloids, they were a poor match for domain structure and an LCD analogous to TIA1; both have amyloid fibrils or disordered precipitates, as elaborated below. been reported to form amyloids (29). Alternatively, it has been hypothesized that the LCD in multidomain proteins can be un- folded (intrinsically disordered) even in the functional form and A that oligomerization might be driven by nonspecific intermo- RNA Recognition Motif 1, 2, and 3 Low-Complexity lecular interactions between several LCDs on different mono- 1 9 77 106 184 214 287288 386 mers (7, 30, 31).
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