Modulation of RNA condensation by the DEAD-box protein eIF4A

Abstract

eTOC Blurb: The RNA helicase eIF4A limits stress granule formation by reducing RNA condensation. Highlights:•! RNA condensates promote intermolecular RNA-RNA interactions at their surfaces! •! eIF4A helicase activity limits the recruitment of RNAs to stress granules in cells! •! eIF4A helicase activity reduces the nucleation of stress granules in cells! •! Recombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner! Keywords: DEAD-box protein, RNA-RNA interactions, stress granule, ribonucleoprotein ! 2 SUMMARY:Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules are proposed to form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is not well understood. In vitro, we demonstrate that the minimization of surface free energy promotes the recruitment and interaction of RNAs on RNA or RNP condensate surfaces. We demonstrate that the ATPase activity of the DEAD-box RNA helicase eIF4A reduces RNA recruitment to RNA condensates in vitro and in cells, as well as limiting stress granule formation. This defines a new function for eIF4A, and potentially other RNA helicases, to limit thermodynamically favored intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function.

A proposed role for the SARS‐CoV‐2 nucleocapsid protein in the formation and regulation of biomolecular condensates

“…Additionally, the features of the SARS-CoV-2 N protein that facilitate interaction with stress granules may also facilitate biomolecular condensation of N protein and genomic RNA during nascent virion formation at the ER-Golgi intermediate compartment (ERGIC) It is important to note that a recent study reported inhibition of SARS-CoV-2 replication in cell culture models treated with multiple translation inhibitors, including the eIF4A inhibitor zotatifin. 37 Inhibition of eIF4A is typically associated with increased stress granule formation 69,70 and can block replication of certain viruses. 71 However, the mechanism by which translation inhibitors suppress SARS-CoV-2 replication was not examined, so it is unclear whether stress granule induction or global suppression of translation (presumably including the production of SARS-CoV-2 proteins) is predominantly responsible for the observed antiviral effect.…”

Protein phase separation and its role in tumorigenesis

“…Another class of proteins linked to cancer are confirmed regulators of SG assembly. For instance, members of RNA-dependent DEAD-box helicases (DDXs) are conserved regulators of RNA-containing condensates like SGs ( Hondele et al, 2019 ; Tauber et al, 2020 ). The ATPase activity of DDXs is required for controlling the partitioning of RNA between different types of condensates.…”

Cytoplasmic mRNPs revisited: Singletons and condensates

“…Another candidate as a general RNA chaperone is the ATP-dependent DEAD-box helicase eIF4A, which decreased RNA condensation in vitro and limited stress granule formation in live cells. [32] We conclude that pre-translational mRNPs appear to be single units absent from biomolecular condensates. A notable exception to this generalization is germ granules, in which stochastic seeding of single mRNPs is followed by multi-copy homotypic clustering in a sequenceindependent manner.…”

“…This inference is based on analysis of mRNA in stress granules, exhibiting additional RNA-RNA interactions in the absence of ribosomes. [31,32] Pre-translational mRNPs containing EJC components, such as Magoh and eIF4A3, were found to behave like flexible rod-like structures or polymers. [6] The study by Metkar The low potential for higher-order mRNA structure is in line with earlier DMS and SHAPE probing studies in vivo.…”