A quantitative live-cell imaging framework is developed that resolves distinct kinetic phases of SG assembly at single-cell resolution and combines these measurements with single-cell analysis of protein synthesis and identifies the conserved eIF4B RRM as a regulator of productive higher-order organization of the translation initiation machinery.
Abstract
Cells respond to environmental stress by rapidly remodeling translation and assembling stress granules (SGs), which are dynamic ribonucleoprotein condensates that contain untranslated mRNAs, translation initiation factors, and 40S ribosomal subunits. Although the translation initiation factor eIF4B has been implicated in SG biology, the contribution of its highly conserved RNA recognition motif (RRM) to SG assembly has remained unclear. Here, we developed a quantitative live-cell imaging framework that resolves distinct kinetic phases of SG assembly at single-cell resolution and combines these measurements with single-cell analysis of protein synthesis. Using this approach, we show that disruption of the eIF4B RRM delays SG nucleation, slows SG assembly, and reduces the number of SGs formed, while having little effect on mature SG size. Biochemical analyses revealed that the RRM mutant retained high-affinity binding to both RNA and the 40S ribosomal subunit and exhibited only a modest reduction in eIF4A helicase stimulation activity but displayed altered RNA engagement, consistent with impaired RNA-dependent organization of the translation initiation machinery. Coupling SG kinetics with single-cell measurements of protein synthesis further revealed that delayed SG nucleation is associated with reduced translational repression during oxidative stress. Together, our findings identify the conserved eIF4B RRM as a regulator of productive higher-order organization of the translation initiation machinery and establish a quantitative framework for investigating how SG assembly and translational remodeling are coordinated during cellular stress.
Stress granules (SGs) are stress-induced ribonucleoprotein condensates assembled around untranslated mRNAs and RNA-binding proteins. G3BP1 is a central regulator of SG formation, yet the molecular events that initiate G3BP1-mediated condensation remain poorly understood. Current models propose that condensation is initiated by RNA–RNA interactions, G3BP1 self-association, or RNA-dependent assembly of G3BP1 into higher-order networks. To define the earliest steps of condensate formation, we employed high-speed atomic force microscopy (HS-AFM) to monitor G3BP1–RNA assembly at the nanometer scale. HS-AFM revealed that G3BP1 first associates with RNA to form discrete nascent assemblies that progressively recruit additional RNA and G3BP1 molecules. These assemblies subsequently grow into higher-order RNA–protein condensates through stepwise assembly. Together, these observations identify RNA-bound G3BP1 assemblies as the initiating structures of condensate formation and provide a framework for understanding the early stages of stress granule assembly.
S. Mahmud, Noriyuki Kodera, Hanae Sato· International Journal of Mol...· 0 citations
It is proposed that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts have evolved structural features that fine-tune protein levels that may be prevalent in bacteria which exhibit uncoupled transcription and translation.
Zachory M. Park, Christina R. Savage, Amanda R. Decker-Farrell et al.· Cell Reports· 0 citations
Stress granules (SGs) are cytoplasmic biomolecular condensates that assemble when translation initiation stalls, sequestering stalled preinitiation complexes and associated RNA-binding proteins. How individual initiation factors are targeted to SGs and released following stress recovery to reinitiate translation remains poorly understood. Here, combining a NEDD8-conjugate proteome with our previously reported arsenite-induced NEDD8 interactome and curated RNA granule databases, we find that eIF3g and eIF3i are shared, high-confidence NEDDylated SG components. NEDDylation of eIF3g and eIF3i-associated complexes is readily detected at steady state and declines under arsenite stress. Intriguingly, only full-length eIF3g is recruited to SGs. eIF3g lacking the RRM domain strongly inhibits SG formation, whereas the RRM domain alone neither inhibits SG assembly nor localizes to SGs. Blocking the NEDD8 pathway— by NAE inhibition with MLN4924, depletion of NEDD8 pathway components, or expression of the deNEDDylase NEDP1—accelerates the loss of eIF3g and eIF3i protein during stress. Our data indicate that NEDDylation marks a degradation-resistant pool of eIF3g/eIF3i that is competent for SG localization, linking the NEDD8 pathway to initiation-factor proteostasis and condensate partitioning, and potentially making these factors available for translation reinitiation during stress recovery.
A. Jayabalan, Ramesh Mariappan, Abirami Rajendiran et al.· bioRxiv· 0 citations
DCP2, encoding a major mRNA decapping enzyme, is identified as a previously unrecognized ISR-induced transcript and it is shown that translational induction of DCP2 depends on an overlapping ORF whose conserved 3′ region, corresponding to a ribosome pausing site, acts as a potent inhibitory element.
Ligand dimerization represents a powerful strategy to enhance avidity, potency, and selectivity. Leveraging the natural-product molecular glue rocaglamide (RocA), we identified BisRoc, a dimeric rocaglate ligand that potently and durably suppresses translation and exhibits greater specificity across a cancer cell line panel than the monomeric RocA. CRISPRi screening revealed that BisRoc activity is influenced by cellular context, including IFITM-mediated uptake, ABC-type efflux transporters, and the translation initiation factor eIF4A2. Mechanistic studies showed that the paralogs eIF4A1 and eIF4A2 are differentially sensitive to BisRoc-induced dimerization. Owing to the presence of multiple binding sites on RNAs, BisRoc-bridged eIF4A-RNA motifs assemble into higher-order complexes that promote stress-granule formation more efficiently than monomeric RocA. Given the widespread multivalency of RNA-RBP interactions, this ligand dimerization strategy may be extended to modulate the higher-order assembly of other RNA-binding proteins.
Jie Liu, Megan K. Moore, Kevin Lou et al.· ACS Central Science· 0 citations
