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

Abstract

Stress 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, TIA1, or FMRP can target mRNAs into SGs in a dose-dependent manner with numerous interactions required for efficient SG partitioning, which suggests individual protein interactions have small effects on the SG partitioning of mRNPs. This 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.

FMRP activates the translation of large autism/intellectual disability proteins and stimulates N-end rule E3 ligase Poe/UBR4 production within puromycin-sensitive RNP particles

“…RNP granules are thought to form as a result of multiple weak interactions between RNA and RNA binding proteins (Matheny et al 2020). In addition, protein interaction domains found in partially translated polypeptides can also direct polysomes to specific subcellular sites (Yanagitani et al 2011).…”

Limited effects of m6A modification on mRNA partitioning into stress granules

“…U-2 OS cells stably expressing 0-and 25-BoxBluciferase RNA was generated by transducing pLenti-EF1-luciferase-blast and pLenti-EFluciferase-25-BoxB-blast. The plasmids were created by inserting luciferase and luciferase 25-BoxB derived from plasmids described in Matheny et al, (2021)…”

“…Single-molecule FISH probes except for the POLR2A (SMF-2006-1, Biosearch Technologies) and firefly luciferase mRNA single-molecule FISH probes (Matheny et al, 2021) are now created using a method as described in Gaspar et al, (2017) (Gaspar et al, 2017). Oligo sequences can be found in Supplemental Table 1.…”

“…Thus, we sought to directly test our model by other means. To test the merit of our linear regression model and experimentally quantify how much YTHDF-m 6 A interaction plays a role in recruiting RNA stress granules, we tethered 25 YTHDF proteins on luciferase reporter RNAs and examine its localization to stress granules using the λN-BoxB system (Matheny et al, 2021). YTHDF1 and 2 fused to GFP-λN transgene are transfected into U-2 OS cells stably expressing a luciferase reporter containing 25-BoxB stem-loops ( Figure 3A).…”

In vivoCGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity

“…It is also possible that interactions of specific factors with CGG repeat RNA may only occur in particular cellular states, such as after activation of cellular stress pathways. Dynamic RNAprotein and RNA-RNA interactions change under cellular stress (Van Treeck & Parker, 2018;Matheny et al, 2020) and repeat RNAs such as ALS/FTD-associated C9ORF72 GGGGCC repeats can partition into stress granules and interact with specific proteins (Fay et al, 2017). Thus, capturing context-specific repeat RNA-protein interactions in vivo might reveal novel modulators of repeat RNA biology and pathogenesis.…”