Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation

Abstract

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a “prion-related domain” or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

On the specificity of protein–protein interactions in the context of disorder

“…Disordered regions are important co-actors and drivers in these processes, and multivalent IDR-IDR contacts are often seen. However, the process is not restricted to IDPs [88][89][90], but more to the property of multivalency (Figure 1D), although disordered linkers as well as IDPs play additional roles in this process [91]. RNA and DNA are inherently highly multivalent, and LLPS phenomena involving proteins typically active in RNA and DNA metabolism are currently some of the most thoroughly described.…”

The Multifunctional Faces of T-Cell Intracellular Antigen 1 in Health and Disease

“…Stress granules are non-membranous aggregates that form from mRNAs arrested at translation initiation and thus contain translation initiation factors, in addition to proteins with both RNA-binding and other functions [58,133] (Figure 5). Similar to other nonmembranous intracellular compartments, such as processing bodies or Cajal bodies, SGs are formed by LLPS [99,134] (Figure 5). This implies that within the cell, the regions change their material properties, which become liquid-like and can be differentiated from the rest of the cytoplasm without the need to be separated by a membrane [134].…”

“…This implies that within the cell, the regions change their material properties, which become liquid-like and can be differentiated from the rest of the cytoplasm without the need to be separated by a membrane [134]. Interestingly, upon some endogenous or exogenous stress situations, the cell can trigger LLPS of specific proteins, which can also include other proteins as part of these condensates [99,100,134]. Proteins with low-complexity, intrinsically disorganized prion-like domains (PLDs)-as is the case of the C-terminal domain of TIA1-are known to have the ability to trigger LLPS.…”

“…Indeed, it has been shown that TIA1 can induce LLPS not only in vivo, associated with RNA and other proteins to form SGs [57,[101][102][103], but also in vitro by self-aggregating in the presence of RNA or single-stranded DNA, for which it has high affinity [100][101][102][103]. Although these C-terminal PLDs of TIA1 are known to interact with one another during SG formation, remaining disorganized, the precise arrangement of the protein within aggregates is unknown, although a model has been proposed whereby TIA1 gives rise to micellar-like structures [99]. While the sequence-intrinsic features that drive LLPS or control the material properties of the resulting aggregates are not yet fully understood, the existence of a "molecular grammar" has recently been demonstrated, according to which interactions between specific amino acids can either promote or impede the process [134].…”

Monitoring protein unfolding transitions by NMR-spectroscopy

“…Intermolecular interactions driving liquid-liquid phase separation (LLPS) are especially difficult to investigate and NMR is one of few valuable methods to gain these molecular details (Emmanouilidis et al 2021). Not only intrinsically disordered proteins but also folded and thermodynamically stable proteins drive LLPS and get analysed by NMR chemical shift analyses (Fritzsching et al 2020). Last but not least, evolution might have started in the deep sea under high pressure conditions, which influences protein stability, LLPS and protein function (Cinar et al 2019;Luong et al 2015).…”