mTORC1 promotes TOP mRNA translation through site-specific phosphorylation of LARP1

Abstract

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5′TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1’s translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 ‘pendular hook’ engages the TOP mRNA 5′-end to repress translation, but only in conditions of mTORC1 inhibition.

Genes with 5′ terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein

“…LARP1 has emerged as a key mediator of mTORC1-mediated inhibition of TOP transcripts (Gentilella et al 2017;Philippe et al 2020;Hong et al 2017;Jia et al 2021;Gentilella et al 2017).…”

“…Finally, to identify potential host factors that may facilitate the translation of TOP transcripts in the presence of Nsp1, we focused on La-related protein 1 (LARP1), a key effector in mTOR mediated regulation of TOP mRNAs (Jia et al 2021;Philippe et al 2020). We used a LARP1 knockout (KO) HEK293T cell line for further reporter assays.…”

“…Additionally, to assess the role of LARP1 in the presence of Nsp1, the above reporter assay was either carried out in HEK293T-Cas9 control cells or LARP1 knockout cell line (KO). Both HEK293T-Cas9 and LARP1 KO cell line were a kind gift from Prof. Tommy Alain, CHEO Research Institute (Jia et al 2021).…”

Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

“…On the other hand, PTMs can affect the activity of RBPs ( Yu, 2011 ; Thapar, 2015 ; Lovci et al, 2016 ). For example, PTMs have been shown to regulate RBPs in diverse cellular contexts, including protein translation ( Imami et al, 2018 ; Jia et al, 2021 ), RNA stability and processing ( Durand et al, 2016 ; Zhang et al, 2018 ), splicing ( Stamm, 2008 ), and phase separation ( Hofweber and Dormann, 2019 ).…”

“…LARP1 might be a good example for the latter case: Upon inhibition of the upstream kinase mTORC1, the abundance of LARP1 in RIC-like experiments increases ( Smith et al, 2020 ), suggesting stronger RNA-binding. While LARP1 interacts in cells with multiple transcripts ( Hong et al, 2017 ), mTORC1-dependent phosphorylation modulates affinity toward the specific group containing the 5′ TOP motif ( Jia et al, 2021 ). Whether 5′ TOP motif RNAs have different isolation/cross-linking efficiency is not known.…”