RNA partitioning into stress granules is based on the summation of multiple interactions

Abstract

SUMMARYStress granules (SGs) are stress-induced RNA-protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple elements. Moreover, artificial tethering of G3BP1 or TIA1 can target mRNAs into SGs in a dose-dependent manner that suggests individual protein interactions have small effects on the SG partitioning of mRNPs, which is supported by the observation that the SG transcriptome is largely unchanged in cell lines lacking the abundant SG RNA-binding proteins G3BP1 and G3BP2. We suggest the targeting of RNPs into SGs is due to a summation of potential RNA-protein, protein-protein, and RNA-RNA interactions with no single interaction dominating RNP recruitment into SGs.

RNP Granule Formation: Lessons from P-Bodies and Stress Granules

“…The observation that NORAD contains many elements promoting SG accumulation and the observation that efficient SG targeting by tethered proteins requires 25 sites of tethering suggest a model wherein the accumulation of a given RNP into a SG is due to the summation of many interactions (Matheny et al 2020). This view is consistent with the strong bias toward longer RNAs in SGs, which by their length would simply have more sites for positive interactions.…”

“…More direct evidence that some RNA-binding proteins can target RNPs into SGs has come from examining how artificial tethering of proteins to reporter RNAs affects their accumulation in SGs. For example, the tethering of G3BP1 or TIA-1 to a luciferase reporter mRNA increases its accumulation in stress granules in a dose-dependent manner (Matheny et al 2020). This provides direct evidence that proteins bound to an RNA can dictate its accumulation in an RNP granule.…”

“…Interestingly, this summative model predicts that any given protein would have small effects on the set of RNAs that accumulate in a SG. This provides an explanation for the surprising observation that U-2 OS cells lacking G3BP1 and G3BP2, two abundant and important proteins for SG formation, form SGs with essentially the same transcriptome as wild-type cells (Matheny et al 2020).…”

“…For example, insertion of the NORAD lncRNA, which is highly enriched in SGs (Khong et al 2017), into a reporter efficiently targets that reporter RNA into SGs (Matheny et al 2020). Moreover, deletion analysis of NORAD demonstrates that multiple regions of NORAD act in an additive manner to promote SG accumulation of the RNA (Matheny et al 2020).The number of binding sites for known SG RBPs within the region of NORAD inserted into the reporter correlates with increased SG enrichment, whereas control RNAs of the same length, but without known sites for RBPs, showed lesser accumulation in SGs (Matheny et al 2020). This suggests that the SG accumulation of NORAD may be largely dictated by the presence of multiple binding sites for SG proteins and raises the possibility that nonspecific RNA-RNA interactions contribute less to the SG accumulation of NORAD RNA.…”

Identification of the stress granule transcriptome via RNA-editing in single cells andin vivo

“…Furthermore, even though a clear enrichment in adenylate-uridylate AU-rich elements (ARE) has been established in ER-stress driven stress granules mRNAs (21), a true recruitment motif has not yet been identified. Recent efforts shows that AREs and Pumilio recognition elements increase mRNA partitioning into stress granules (25). However valuable, these techniques are somewhat limited by the step of cell fractionation and stress granules purification.…”