Fig 2: Two different downstream targets of hnRNPK during myogenic differentiation. During myogenic differentiation, hnRNPK has two different downstream targets. One is myogenin, which codes for a regulator of myogenic differentiation. hnRNPK represses the myogenin expression by binding to the ccawmcc motifs on Myoparr. The other target is aminoacyl-tRNA synthetases. hnRNPK regulates the expression of aminoacyl-tRNA synthetases via the eIF2a/Atf4 pathway. In hnRNPK-depleted cells, the hyperactivated expression of these genes may lead to the locally spherical formation of myotubes.

Fig 3: ATF4 is both necessary and sufficient for insulin-mTORC1 signaling to induce select gene targets. (A) Immunoblot analysis of primary mouse hepatocytes transfected with control (siCT) or Atf4-targeting siRNAs followed by overnight serum starvation and treatment with 100 nM insulin for 8 h. (B) Gene expression in cells treated as in (A) plotted as mean ± SEM relative to the siCT-transfected serum-starved cells (n = 3 independent experiments). (C) Immunoblot analysis of Atf4fl/fl and LAtf4KO primary mouse hepatocytes serum-starved overnight, and subsequently, treated with 100 nM insulin for 6 h following a 30-min pretreatment with vehicle (DMSO) or 20 nM rapamycin. (D) Gene expression in cells treated as in (C) plotted as mean ± SEM relative to the Atf4fl/fl serum-starved cells (n = 3). (E) Immunoblot analysis of Atf4fl/fl and LAtf4KO primary mouse hepatocytes infected with AdGFP or AdATF4 (MOI = 10) for 24 h. (F) Gene expression from a representative experiment of cells treated as in (E) plotted as mean ± SEM relative to the Atf4fl/fl AdGFP sample (n = 3). (G) Immunoblot analysis of WT primary mouse hepatocytes infected with AdGFP or AdATF4 (MOI = 10) for 24 h followed by treatment with vehicle (DMSO) or 20 nM rapamycin for 8 h. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA (B,D) or one-way ANOVA (F)).

Fig 4: hnRNPK regulates the expression of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. (A) A Metascape analysis of genes regulated by Myoparr KD and hnRNPK KD. The red frame indicates the GO terms that were specifically enriched in hnRNPK KD. (B–G) qRT-PCR to detect the expression of Aars (B), Gars (C), Iars (D), Nars (E), Sars (F), and Atf4 (G) 48 h after either Myoparr KD or hnRNPK KD. n = 3, mean ± SD. ** p < 0.01, * p < 0.05, n.s., not significant. (H,I) Western blot showing the increased expression of Atf4 in differentiating C2C12 cells (H) and mouse primary myoblasts (I) 48 h after hnRNPK KD. The tubulin expression served as an internal control. Blots are representative of three experiments.

Fig 5: Feeding and insulin induce hepatic ATF4 and established gene targets by mTORC1 signaling. (A and B) Eight-week-old male mice were fasted for 12 h and refed with a high carbohydrate diet for 6 h following pretreatment with vehicle or 10 mg/kg rapamycin (n = 6/group). (A) Liver lysates were immunoblotted to assess mTORC1 signaling and ATF4, with mean ATF4 protein to tubulin quantification plotted ± SEM and normalized to the fasted group (right). (B) Liver gene expression plotted as mean ± SEM relative to the fasted group. (C) Immunoblot analysis of primary mouse hepatocytes serum-starved overnight and treated with 100 nM insulin following a 30-min pretreatment with vehicle (DMSO), 20 nM rapamycin, or 750 nM Torin1 for the indicated time points. (D) Immunoblot analysis of primary mouse hepatocytes transfected with control (siCT) or a combination of Eif4ebp1 (4ebp1) and Eif4ebp2 (4ebp2) siRNAs, followed by overnight serum starvation and treatment for 8 h with 100 nM insulin following a 30-min pretreatment with vehicle, 20 nM rapamycin or 500 nm Torin1. (E) Immunoblot analysis of primary mouse hepatocytes treated for the indicated time points as in (C). (F) Gene expression in cells treated as in (E) plotted as mean ± SEM relative to serum-starved cells (n = 4 independent experiments). (G) Immunoblot analysis of primary human hepatocytes serum-starved overnight, and subsequently, treated with 100 nM insulin following a 30-min pretreatment with vehicle (DMSO), 20 nM rapamycin, or 500 nM Torin1 for the indicated time points. (H) Gene expression in cells treated as in (G) plotted as mean ± SEM (n = 3) relative to the serum-starved cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA).