Fig 2: Analysis of MTR4 KOW-NVL complex by NMR and site directed mutagenesis. a Overlay of 1H-15N-HSQCs of either MTR4 KOW alone (black) or in complex with a six-fold excess of NVL2 (red). Selected residues experiencing large chemical shift perturbations (CSPs) are labeled. b Plot of CSPs per residue of the MTR4 KOW sequence. The red line marks the threshold of significant CSPs, which are mapped on the model of MTR4 KOW (PDB 6IEH) in panel c. Gaps indicate either prolines or residues that could not be assigned. c MTR4 KOW domain (PDB 6IEH) with labeled secondary structural elements as assigned by NMR and residues showing significant CSPs (displayed as red/green spheres). d Protein co-precipitations by pull down assays. GST-tagged MTR4∆N (WT or mutants) were incubated with Trx-NVL167–216 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–4) and 30% of the eluates (lanes 5–8) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue

Fig 3: SKIV2L2 mediates miR-155 regulation of circHIF1α expression. (A) CFs were transfected with pAd-SKIV2L2 and then exposed to normoxia or hypoxia for 24 h. circHIF1α expression was detected by qRT-PCR. *P < 0.05 vs. their corresponding control. (B) CFs were transfected with si-SKIV2L2 and then exposed to normoxia or hypoxia for 24 h. circHIF1α expression was detected by qRT-PCR. *P < 0.05 vs. si-con + hypoxia. (C) circMyh9, circFOXO1 and circDnmt3a expression in CFs treated as in (B) was detected by qRT-PCR. **P < 0.01 and ***P < 0.001 vs. si-con. (D) A schematic of HIF1α exons 2 through 8 (top). CF lysates were immunoprecipitated with anti-SKIV2L2, and the immunoprecipitates were detected by qRT-PCR with primers specific for the CS1-6 sequences (bottom). **P < 0.01. (E) An oligo pull-down assay was done with CF lysates and biotinylated double-stranded oligonucleotide (∼150 nt) surrounding and in the CS4 of HIF1α pre-mRNA. The oligo-bound protein was detected by Western blot with anti-SKIV2L2 antibody.**P < 0.01.(F) qRT-PCR detected the enrichment of the CS4 fragment by anti-SKIV2L2 in miR-155−/− and miR-155wt CFs. ***P < 0.001 vs. miR-155wt (G) CFs were transfected with the ASO of the CS4 and pAd-SKIV2L2 for 24 h. circHIF1α expression was detected by qRT-PCR. *P < 0.05 vs. Con, **P < 0.01 vs. ASO. (H) miR-155−/− CFs were transfected with with the ASO of the CS4 and then exposed to normoxia or hypoxia for 24 h. circHIF1α expression was detected by qRT-PCR, **P < 0.01 and ***P < 0.001 vs. miR-155-/-. Data shown in (A)-(H) are mean ± SEM, n = 3 independent experiments.

Fig 4: Analysis of the ZCCHC8-MTR4 KOW complex by site directed mutagenesis. a Sequence alignment of the region between the predicted coiled coil domain and the zinc finger of ZCCHC8 from representative metazoan species, Homo sapiens (Hs), Gallus gallus (Gg), Xenopus laevis (Xl), highlighting NVL-like region (red), Nop53-like AIM (C-AIM) and ZCCHC8 specific I-AIM (purple boxes). The sequences were obtained from the UniProt database and aligned using the T-coffee server59. b Protein co-precipitations by pull down assays testing ZCCHC8 91–211 mutants for MTR4∆N binding ability. GST-tagged MTR4∆N was incubated with either ZCCHC891–211 WT or mutants before co-precipitation with glutathione sepharose beads. A total of 3% of the input (top) and 30% of the eluates (bottom) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Protein co-precipitations by pull down assays testing MTR4∆N mutants for their ZCCHC8 91–211 binding ability. GST-tagged MTR4∆N (WT or mutant variants) were incubated with MBP-ZCCHC8 91–211 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes, 1–4) and 30% of the eluates (5–8) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. d Cellular co-IP assay. FLAG-tagged ZCCHC8 constructs (WT/IF mutant/CTD deletion) were transiently expressed in cells stably expressing MTR4-LAP. After precipitation of MTR4 taking advantage of the LAP tag, a total of 0.5% of the input (left) and 8.0% of the eluates (right) were analyzed on 4–12% SDS-PAGE gel followed by western blotting analysis. The primary antibody used is indicated below the panel

Fig 5: Ectopic expression of GLUT1 rescued the defective tumorigenesis of HCC cells after MTR4 silencing.a Ectopic expression of GLUT1 in control and MTR4 KD cells was analyzed for MTR4 and GLUT1 by western blotting. Representative data from two independent experiments are shown. b, c ECAR in control cells and MTR4 KD cells transfected with either empty vector (EV) or plasmids expressing GLUT1 (GLUT1 OE) in response to glucose, oligomycin, and 2-DG. Data are presented as mean value ± s.d., two-way ANOVA with a Tukey’s multiple comparison test. n = 3 independent experiments for each group. d The proliferation of indicated cells were analyzed with CCK8 assay. Data are presented as mean value ± s.d. Two-way ANOVA with a Tukey’s multiple comparison test. n = 3 independent biological samples. e The weight of tumors formed by indicated cells in NSG mice was measured and compared. Data are presented as mean value ± s.d. One-way ANOVA, followed by Bonferroni post-tests. n = 8 independent biological samples for each group. p value is indicated. Source data are provided as a Source Data file.