Fig 2: Comparison of relative mRNA expression ratio (PTC/matched normal thyroid tissue) and protein expression for A-RAF (A), B-RAF (B), C-RAF (C), and COT (D). Relative expression was calculated using the StepOne™ Real-time PCR System (Applied Biosystems, Foster City, CA, USA). 2ΔCtn = 2CtGAPDH-Cttarget calculation was used for relative mRNA expression value. Relative mRNA expression ratio was calculated by relative mRNA expression value of PTC/that of matched normal thyroid tissue (n = 135). Average ratios were compared with the paired t test. Data are average mean values. ∗∗P < 0.01, ∗∗∗P < 0.001. All P values are 2-sided. (E) Representative western blot analysis to compare protein expression between PTC and matched normal thyroid tissues (n = 3). All experiments were repeated 3 times, and each experiment was performed in triplicate. COT = Cancer Osaka Thyroid Oncogene mitogen-activated protein kinase kinase kinase 8, PTC = papillary thyroid cancer.

Fig 3: Model for the role of hnRNP H/F in regulating mRNA translation in GBM. Increased expression of hnRNP H/F in GBM [26] indirectly (i) and/or directly (ii) regulates mRNA translation through different overlapping mechanisms, including: (i) A-Raf mRNA splicing that in turn modulates the MEK-ERK/MAPK pathway to control the phosphorylation of eIF4E (this work) known to impact on translation, and (ii) the direct involvement of hnRNP H/F in translation of specific mRNAs [26]. The underlying regulatory mechanisms involve modulation of RG4 folding by hnRNP H/F and result in both global and selective control of translation impacting key functional cellular pathways, such as cell migration, proliferation and DNA repair, involved in GBM tumor growth and resistance to treatments.

Fig 4: RG4 stabilization affects A-Raf splicing and eIF4E phosphorylation. (A) Immunoprecipitation (IP) of RNA–protein complexes (RIP) using control IgG (IP IgG) or BG4 (IP BG4) antibody, on cytoplasmic extracts from U87 cells untreated (NT) or treated with 20 µM carboxypyridostatin (cPDS) for 2 h and followed by RT–qPCR analysis of A-Raf, USP1 and HPRT mRNAs. Data are plotted as mean values ± SEM of n = 3 independent experiments. (B) Quantitative RT-qPCR using specific primers for A-Raf short isoform on total RNA extracted from LN18 cells treated with dose scale of carboxypyridostatin (cPDS) for 4 h. A-Raf short mRNA levels were normalized against GAPDH and data were plotted relatively to the non-treated condition (0 µM cPDS). Data are presented as mean values ± SEM of n = 3 independent experiments. (C) Western blot analysis of A-Raf short, phospho-ERK 1/2 (p-ERK 1/2), ERK 1/2, phospho-eIF4E (p-eIF4E) and eIF4E expression in LN18 cells treated with dose scale of carboxypyridostatin (cPDS) for 48 h. Shown is a representative result from n = 3 independent experiments. (D) A-Raf short, p-ERK 1/2 and p-eIF4E protein levels in (C) were normalized to total GAPDH, ERK 1/2 and eIF4E protein levels, respectively, and plotted relatively to the non-treated condition (0 µM cPDS). Data are presented as mean values ± SEM of n = 3 independent experiments. For panels (A,B,D): red and dark red bars represent increasing concentrations of cPDS; * p < 0.05; ** p < 0.005; NS—non-significant (two-sided paired t-test).

Fig 5: C-Raf is regulated by the proteasome through the CTLH complex. Non-targeting shRNA control and shRNA RanBPM cells were treated with 10 μM MG132 or DMSO, as vehicle, for 24 h. RIPA buffered whole cell extracts of HeLa (A), and HCT116 (B) were analyzed by Western blot with RanBPM, c-Raf and β-actin antibodies to detect RanBPM, c-Raf and β-actin proteins, respectively. c-Raf protein levels were normalized to β-actin levels. Quantifications of relative c-Raf protein levels are shown with error bars indicating SD (n = 4). * p < 0.05; ** p < 0.01; ns, no significance. (C) c-Raf is ubiquitinated in a CTLH complex-dependent manner. Hela control (WT Hela) and RMND5A KO cells were transfected with GST-ΔN-c-Raf and/or HA-Ubiquitin as indicated. GST pull-downs performed using whole cell extracts were analyzed by Western blot with the indicated antibodies. Input represent 5% of the extracts used for pull-down. The asterix (*) indicates a non-specific band (NS). (D) A-Raf and B-Raf expression is increased in RMND5A KO cells. Extracts from HEK293 control and RMND5A CRISPR KO cells were analyzed by Western blot with the indicated antibodies. Quantifications are shown below with error bar indicating SD, n = 8 for A-Raf, **** p < 0.0001 and n = 3 for B-Raf, ** p < 0.01.