Fig 1: HIFAL translocates into the nucleus via binding with hnRNPF.a, b HIAFL fragment nt 240–300 is essential for its nucleus translocation. a Various HIFAL fragments were transfected into HIFAL null MDA-MB-231 cells. RNA in the cytoplasmic or nuclear fractions was isolated for HIFAL detection. P values were determined Ordinary one-way ANOVA with Dunnett’s post hoc test. Bar graphs represent means ± SD of fifteen random fields. b Deletion of HIFAL fragment nt 240–300 abrogates its translocation into the nucleus upon hypoxia. The biotin-labeled exogenous wild type or mutated HIFAL without nt 240–300 was transfected into HIFAL null MDA-MB-231 cells, and detected by FITC labeled anti-biotin antibody. Scale bars, 10 µM. c Wildtype but not mutated HIFAL binds to hnRNPF, as detected by mass spectrum identifications. The peptide number of hnRNPF or hnRNPK detected by mass spectrum was shown in the table (lower). d The region for HIFAL nuclear translocation contains similar RNA sequence with SIRLOIN. The conserved sequence of SIRLOIN or HIFAL nt 240–300 was highlighted in red. e, f HnRNPF binds with WT but not mutated HIFAL without the nuclear translocation region (nt 240–300), as detected by RNA pulldown (e) and RNA immunoprecipitation assay (f). Bar graphs represent means ± SD of experimental triplicates. P values were determined by two-sided unpaired t-test. g Binding of PKM2 or PHD3 with hnRNPF depends on HIFAL. The immunoprecipitation of hnRNPF was performed in HIFAL WT or null MDA-MB-231 cells. h, Hypoxia induces hnRNPF overexpression and nuclear translocation. i Knockdown of hnRNPF abolishes HIFAL nuclear translocation. HIFAL (green) was detected by digoxin labeled probes. Scale bars, 10 µM. j Diagram of the full length hnRNPF and its serial truncated mutants. k The RRM truncated hnRNPF mutant retains the ability of binding with HIFAL. Full length or the RRM deletion mutant (D RRM) of hnRNPF was transfected into HEK293 cells for HIFAL RNA-pull down assay. l, m HnRNPF 141–178aa region contains the HIFAL binding domain. n hnRNPF mutant without the HIFAL binding-domain cannot induce the nuclear translocation of HIFAL. The hnRNPF null cells were transiently transfected with hnRNPF WT or D6 mutant and cultured under hypoxia. Scale bars, 10 µM. Source data are provided as a Source Data file.