Figure 3. Dynamic rearrangement of LARP1 biding to the UTRs of RpL32 mRNA is regulated by the phosphorylation of LARP1.
(A) The effect of amino acid and mTOR inhibitor on the levels of endogenous LARP1 binding to the 5’ and 3’UTR of RpL32 mRNA. HEK293T cells were transfected with the indicated reporter mRNAs. Endogenous LARP1 was IPed, and the levels of co-IPed luciferase mRNA were determined by qPCR. Data were normalized by input luciferase mRNAs and the amount of IPed LARP1. *p<0.05, mean±SEM (n = 3). (B) LARP1 phosphorylation by mTOR and S6K1/Akt induces its dissociation from the PES in the 5’UTR of RpL32 mRNA. The wild type and the LARP1 4A mutant were transfected with the indicated reporter mRNAs. Data were expressed as Figure 3A. N.S. denotes ‘not significant’. *p<0.05, mean±SEM (n = 3). (C) The effect of alanine substitutions of all the phosphorylation sites of LARP1 identified in this study on the binding to the 5’UTR of RpL32 mRNA under growth conditions. N.S. denotes ‘not significant’. *p<0.05, mean±SEM (n = 3). (D–E) Both mTOR- and S6K1/Akt-dependent LARP1 phosphorylation are necessary for its dissociation from the 5’UTR of RpL32 mRNA, while either mTOR or S6K1/Akt phosphorylation of LARP1 is sufficient to maintain its binding to the 3’UTR. The wild type and the indicated LARP1 mutants were IPed, and the levels of co-IPed 5’ or 3’ reporter mRNA were determined by qPCR (D). HEK293T cells were starved with amino acids or treated with the indicated inhibitors for 1 hr, and levels of co-IPed 5’ or 3’ reporter mRNA with endogenous LARP1 were determined (E). Data were normalized by input luciferase mRNAs and the amount of IPed LARP1. *p<0.05, mean±SEM (n = 3). (F) LARP1 constitutively interacts with the RpL32 reporter RNA containing both the 5’ and 3’ UTRs in a manner independent of the PES in the 5’UTR and mTOR activity. Endogenous LARP1 PAR-CLIP was performed in the presence or absence of Torin1 treatment. Levels of LARP1-bound reporter mRNA were determined by qPCR. Data were normalized by input luciferase mRNAs and the amount of IPed LARP1. N.S. denotes ‘not significant’, mean±SEM (n = 3). (G) The PES motif on 5’UTR of RpL32 is the cis-acting element necessary for translation inhibition in response to mTORC1 inhibition. HEK293T cells were transfected with the indicated reporter mRNAs and grown in normal growth media (10% serum) or mild starvation media (1% serum). After 24 hr, luciferase activity was measured and normalized by luciferase mRNA levels. *p<0.05, mean±SEM (n = 3). (H) Phosphorylation of LARP1 and its dissociation from the 5’UTR are critical for the translation of RpL32. HEK293T cells lacking endogenous LARP1 were transfected with the indicated shRNA-resistant LARPs and reporter mRNAs. 48 hr post-transfection, luciferase activity was measured and normalized by luciferase mRNA levels (left panel). *p<0.05, mean±SEM (n = 3). Levels of transfected Myc-LARP1s were shown (right panel). (I) Phosphorylated LARP1 plays a positive role in ribosomal protein translation. Myc-tagged wild type LARP1 and the 4A mutant LARP1 were stably expressed in HEK293T cells by retrovirus-mediated infection to achieve lower levels of LARP1 expression, and endogenous LARP1 was knockdown by LARP1 shRNA targeting its 5’UTR. Levels of RP proteins were determined by western blotting and the intensity of the bands was quantified.