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A novel RRM activity orchestrates ribosome homeostasis

James A. SabaiD, Phoebe E. WhiteiD, Alexander Maxwell BurroughsiD, Lakshminarayanan AravindiD, Rachel GreeniD

Abstract

Ribosome concentration varies across cell type, state, and disease, yet the mechanism by which mammalian cells sense ribosome levels to regulate ribosome synthesis remains unknown. Previous studies implicate LARP1 in binding ribosomes and, separately, in repressing mRNAs containing TOP motifs (TOPs), which encode all ribosomal proteins as well as several translation factors. Here, we show that ribosome binding is the essential licensing event for LARP1-mediated TOP repression. Phylogenetic analysis reveals that LARP1's ribosome binding region is part of a highly conserved RNA recognition motif (RRM) which is predicted to directly interact with its TOP-binding HEAT repeat domain. Ribosome binding activates the HEAT repeats to bind and repress TOPs, thereby repressing TOP translation and ribosome synthesis. Mutants which uncouple the coordinated functions of these domains constitutively repress TOPs and compromise cell fitness. These data reveal an unprecedented ribosome sensing function of LARP1, orchestrated through the unique coordinating role of its RRM, unveiling the mechanism by which mammalian cells tune ribosome synthesis to demand across diverse contexts.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (10 Sept 2026) · CC BY 4.0

40S subunit binding to a previously unrecognised RRM in LARP1 activates its TOP-binding HEAT repeats, licensing repression of ribosomal protein mRNA translation in human cells.

How mammalian cells sense ribosome levels to tune ribosome synthesis was unknown; earlier work linked LARP1 to ribosome binding and, separately, to repression of TOP mRNAs. This paper proposes that the two activities are coupled through a hidden RRM, with 40S binding licensing TOP repression.

Key results

  1. SuggestedThe region between LARP1's La-wHTH and HEAT repeats is a divergent RRM (RBR-RRM) containing the 40S-binding helix. The three-domain LARP1 architecture is inferred to date to the last eukaryotic common ancestor.

    AlphaFold3 models a 4-stranded beta sheet plus two helices; structure searches hit RRMs (e.g. TM-score 0.67 for human LARP1 to HTATSF1 RRM2; 0.60 for B. floridae LARP1 to Drosophila SNF RRM1). Homologs found across animals, plants, amoebozoans and Metamonada. · Evolutionary conservation of the region spanning…

  2. SuggestedAlphaFold3 predicts the RBR-RRM, with its beta sheet, holds the 40S-binding helix and the HEAT repeats together. Deleting beta-sheet strands in silico removes the predicted helix-HEAT association.

    Low predicted aligned error between RBR-RRM and HEAT repeats in wild type; high PAE for Δ643-648 and Δ832-836. Nine H-bond contacts and one pi-stacking contact listed. Computational prediction only. · LARP1 structural prediction

  3. ShownMutations that break the beta sheet make LARP1 repress a TOP reporter constitutively (insensitive to Torin1); the β1-P mutant also pulls down more 40S subunits. The repression needs functional HEAT repeats.

    β1-P, β1-del and β3-del all gave strongly increased TOP-reporter repression versus WT in LARP1-KO HEK293T cells; the β1-P/HEAT double mutant did not repress. β1-P co-IPed more 40S than WT with and without Torin1. Three independent biological replicates per reporter assay; co-IP blots from at least two replicates. · Disrupting the predicted RBR-RRM/HEAT scaffold…

  4. Shown40S binding is required for LARP1-mediated TOP repression. A 40S-binding-deficient mutant (RBRmut-2) behaves like the HEAT mutant, and a beta-sheet mutation bypasses the defect.

    RBRmut-2 failed to repress the TOP reporter even with Torin1; RBRmut-2/β1-P double mutant restored repression. Phospho-null and phospho-mimetic backgrounds with RBRmut-2 were derepressed to LARP1-KO levels. An earlier mutant (RBRmut-1) had also disrupted the scaffold. · Ribosome binding is required for TOP repression…; LARP1 phosphorylation antagonizes 40S binding

  5. ShownIn vitro, 40S subunits stimulate LARP1 binding to a TOP oligo, and TOP oligo stimulates 40S binding, but non-TOP oligo does neither. In cells, β1-P shifts endogenous TOPs to sub-polysomes and slows growth.

    Dose-responsive 40S stimulation was lost in RBRmut-2, β1-P and HEAT-mut variants. RBRmut-2 shifted TOPs into polysomes. Doubling time was ~40 h for β1-P versus ~24 h for GFP or WT LARP1. · LARP1 couples 40S binding and TOP binding in…; Disrupting the predicted RBR-RRM/HEAT scaffold…

Methods, data and reproducibility

AlphaFold3 modelling and sequence/structure homology searches identified the RBR-RRM and guided LARP1 mutants. These were tested in LARP1-KO HEK293T cells (nanoluciferase TOP reporters, co-IP of 40S proteins, polysome RT-qPCR, growth assays) and with purified components in vitro.

  • LARP1-KO and wild-type HEK293T cells transfected with LARP1 variants; reporter, co-IP, sucrose gradient, qPCR and growth experiments · human (HEK293T, ATCC CRL-3216) · n = 3 biological replicates (reporter, gradient, growth); at least 2 for co-IPs
  • Purified FLAG-LARP1 (574-1096) variants, 40S subunits from HEK293T cells, and 32P-labelled capped TOP / non-TOP 12-mer oligos for in vitro binding · human (HEK293T-derived proteins and subunits) · n = at least two independent experiments; three for TOP-stimulated 40S binding
  • LARP1 homolog sequences across eukaryotes searched against NCBI nr for phylogeny and domain architecture · multiple eukaryotic species
Methods & tools
AlphaFold3, AlphaFold2, PSI-BLAST, MAFFT, DALIlite, Foldseek, FastTree 2.1, ChimeraX 1.9, TM-align, nLuc-PEST TOP/non-TOP reporters, R (ggplot2, emmeans, bio3d), ImageJ
Reagents & environment
ChimeraX version 1.9 and FastTree 2.1 are named; no containers or requirement files stated.
Reproducibility gaps
No data deposited in external repositories and no code released; custom R scripts are not provided. Materials are available on request to the corresponding author.
Limitations (3)
  • The mechanism by which 40S binding activates the HEAT repeats is inferred from AlphaFold3 predictions and clash analysis; the exact biophysical mechanism is not known and no experimental structure of the scaffold is presented. · stated by the authors
  • Differences in 40S binding between dephosphorylated and phosphorylated LARP1 were not captured when cells are translationally active and free 40S subunits are scarce. · stated by the authors
  • Cell experiments use transfected LARP1 variants in LARP1-KO HEK293T cells only, and in vitro work uses a truncated LARP1 (574-1096) with short oligos; physiological relevance in other cell types is untested. · noted in this summary

Who should read:Read this if you study translational control of ribosomal protein mRNAs, LARP proteins, or how AlphaFold3 predictions can guide separation-of-function mutants.

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