The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.DOI: http://dx.doi.org/10.7554/eLife.24146.001

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.

Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs). Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) as ligands. The capacity of chemokine release was determined by immunoassays. Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L. Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine.

The mammalian target of rapamycin complex 1 (mTORC1) controls critical cellular functions such as protein synthesis, lipid metabolism, protein turnover and ribosome biogenesis through the phosphorylation of multiple substrates. In this study, we examined the phosphorylation of a recently identified target of mTORC1: La-related protein 1 (LARP1), a member of the LARP superfamily. Previously, we and others have shown that LARP1 plays an important role in repressing TOP mRNA translation downstream of mTORC1. LARP1 binds the 7methylguanosine triphosphate (m 7 Gppp) cap moiety and the adjacent 5'terminal oligopyrimidine (5'TOP) motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 plays a critical role in the control of TOP mRNA translation via LARP1 but the precise mechanism by which this occurs is incompletely understood. The data described herein help to elucidate this process. Specifically, it show that: (i) mTORC1 interacts with LARP1, but not other LARP superfamily members, via the C-terminal region that comprises the DM15 domain, (ii) mTORC1 pathway controls the phosphorylation of multiple (up to 26) serine and threonine residues on LARP1 in vivo, (iii) mTORC1 regulates the binding of LARP1 to TOP mRNAs and (iv) phosphorylation of S689 by mTORC1 is particularly important for the association of the DM15 domain of LARP1 with the 5'UTR of RPS6 TOP mRNA. These data reveal LARP1 as a major substrate of mTORC1. 1 The biochemical significance of each of these mTORC1-mediated phosphorylation events is reviewed in detail in reference number 25. Fig. 1A). In mammals, the LARP superfamily is comprised by 7 members: La also known as genuine La (the founding member of this superfamily), LARP1, LARP2 (also known as LARP1b), LARP4, LARP5 (also referred to as LARP4b), LARP6 and LARP7 (25,51,52). We have previously shown that LARP1 interacts with mTORC1 via the scaffolding protein RAPTOR (short for regulatory associated protein of mTOR), specifically through contact points with the RNC (RAPTOR N-terminal conserved) region and the WD40 (tryptophan/aspartate repeats 40 amino acids-long) domain (19). Given the high level of sequence conservation between human LARP1 and human LARP2 proteins (60% identity and 73% similarity at amino acid level 2 ), we asked whether LARP2 also interacted with mTORC1. HEK293T cells were transiently transfected with FLAG-tagged human LARP1, human LARP2 or a variant of human LARP2 that lacks the C-terminal region (human LARP2 ∆CT), and subsequently stimulated transfected cells with complete growth media (see experimental procedures section for details). Cells were then lysed and lysates subjected to immunoprecipitation with anti-FLAG tag antibody. FLAG-immunoprecipitates were probed for endogenous RAPTOR and endogenous mTOR proteins by SDS-PAGE/Western blot. The resulting data (shown in Fig. 1B) confirmed our earlier observation (19) that LARP1 (in this particular case exogenous LARP1) interacts strongly with both RAPTOR and mTOR; this interaction is visualized...