Tandem RNA binding sites induce self-association of the stress granule marker protein TIA-1

Abstract

TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3′ UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.

Binding patterns of RNA-binding proteins to repeat-derived RNA sequences reveal putative functional RNA elements

“…We also predicted that these fragments could form stable secondary RNA structures. Notably, both the stoichiometry of RBP-binding sites and RNA secondary structures could be essential for LLPS ( 72 , 73 ). Therefore, our data provide promising candidates for functional RNA elements derived from TE sequences.…”

TDP-43 condensation properties specify its RNA binding and regulation

“…It has been shown that RNAs can have specific effects on condensation properties of RBPs (Loughlin et al, 2021;Maharana et al, 2018;Mann et al, 2019;Sanders et al, 2020), but whether these effects are mediated by full-length RNA molecules or by specific binding regions has remained unclear. Several studies of splicing regulation provide evidence that variable condensation of RNPs might be able to selectively modulate RBP activities at subsets of RNA-binding sites (Ule and Blencowe, 2019).…”

Post-translational Control of RNA-Binding Proteins and Disease-Related Dysregulation

“…The total interaction strength provided by the network of multivalent contacts between proteins and/or RNA is high enough to promote LLPS while ensuring reversible associations and great mobility inside condensates (Strom and Brangwynne, 2019). RBPs can contribute to phase separation by using both wellstructured RBDs and IDRs for binding with RNA molecules, which act as scaffolds during condensation and determine the physicochemical and material properties of the resulting MLO (Drino and Schaefer, 2018;Ryan and Fawzi, 2019;Loughlin et al, 2021). IDRs from RBPs are also involved in protein-protein interactions, including self-association, thanks to the high proportion and distribution pattern of particular residues in their sequences, often clustered in repetitive low-complexity regions (LCRs), such as the arginine/glycine-rich (RGG/RG) boxes and the glutamine/asparagine-rich prion-like domains (PLDs) (Darling et al, 2018;Ryan and Fawzi, 2019).…”

“…Structural details on the binding of TIA-1 to RNA have been thoroughly determined (Aroca et al, 2011;Bauer et al, 2012;Cruz-Gallardo et al, 2013Wang et al, 2014;Waris et al, 2017;Loughlin et al, 2021), although very little information is available about the effect of PTMs on this ARE-RBP. Nevertheless, it is well-known that phosphorylation of TIA-1 and its homolog TIA-1-related protein (TIAR) modulates their activity in the alternative splicing of the Fas receptor.…”