Fig 2: Validation of the correlation between succinylation regulators and m6A regulators at protein level. (A) The expression pattern of CPT1A, KAT2A, SIRT5, SIRT7, LRPPRC, and EIF3B between ccRCC tissues and corresponding normal tissues by immunohistochemistry. Error bars show standard error of the mean, and the middle bar represents the median expression level of each molecule. (B) The correlation analysis between CPT1A and LRPPRC, SIRT5 and EIF3B, CPT1A and EIF3B, SIRT5 and LRPPRC according to immunohistochemistry scores in 42 ccRCC tissues. (C) In ACHN cells, the expression of LRPPRC and EIF3B were detected by western blotting after transient knockdown of CPT1A and SIRT5, respectively.

Fig 3: Lack of feedback between translatomes results in their imbalance and leads to the activation of proteostasis genes. A Comparison of relative synthesis levels for cytosolic-translated OXPHOS subunits with and without 4 h of chloramphenicol (CAP) treatment (100 µg/mL) to inhibit mitochondrial translation. B Relative synthesis for mitochondrial-encoded OXPHOS subunits in LRPPRCKO cells compared to a reconstituted cell line (LRPPRC rescue), expressing LRPPRC in the LRPPRCKO background. C Relative synthesis comparison as in (B) but for nuclear-encoded OXPHOS subunits. D Average synthesis of mtDNA-encoded subunits (tpm/100) compared to average synthesis of nDNA-encoded core subunits (tpm) for each complex in wild-type HEK293T cells, LRPPRCKO cells, and LRPPRC rescue cells. E Average RNA abundance of mtDNA-encoded subunits (tpm/100) compared to average RNA abundance of nDNA-encoded core subunits (tpm) for each complex in LRPPRC cell lines. Error bars throughout show range in averages across replicates, and all panels show Pearson correlation, r. F GO term enrichment analysis of significantly (adjusted p-value < 0.05) differentially expressed genes that show a minimum twofold increase in the LRPPRCKO cells compared to the LRPPRC rescue cell line. Shown are the GO terms after filtering for redundancy. The size of points represents the number of genes in each GO term. p adjust = adjusted p-value. TF = transcription factor. G Cell toxicity graph after 72-h bortezomib treatment. HEK293T WT, LRPPRCKO, or LRPPRC rescue cells were treated with increasing amounts of bortezomib or the vehicle ethanol (EtOH). Cell death was measured via a fluorescence dye and living cells by indirect measurement of ATP levels via luminescence. Shown are the mean relative fluorescence arbitrary units (a.u.) normalized to the respective luminescence of 3 technical replicates, with WT ethanol treated having 2 technical replicates. The range bars represent the standard deviation. The stars represent significant p-values, determined with a Welch two-sample one-sided (less) t test, * signifies p-value < 0.05, ** signifies p-value < 0.01 and *** signifies p-value < 0.001. Two other biological replicates are shown in Additional file 1: Figure S4L. H Left panel shows the immunoblot analysis using whole cell lysates from HEK293 WT, LRPPRCKO, or LRPPRC rescue cells probed for antibodies of markers of the ER and mitochondrial UPR pathways. Bar graphs show signals normalized by ß-ACTIN values and compared to WT to determine fold changes. IRE1 levels are shown as ratios of its phosphorylated form (pIRE1) compared to total (IRE1). Error bars indicate standard deviation between two replicates. T test was used to test for significant changes. * signifies p-value < 0.05, ** signifies p-value < 0.01, and *** signifies p-value < 0.001

Fig 4: LRPPRC-induced upregulation of circANKHD1 regulates the expression of FOXM1 by reducing the inhibitory effect of miR-671-5p and miR-507 via sponge activity. (A) Clustered heatmap for the circRNA expression profiles of T24 cells transfected with LRPPRC-sh2 and sh-NC. The circRNAs are classified according to Pearson correlation analysis. The numerical data represent the serial number of circRNAs in circBase. (B) qRT-PCR for circANKHD1 in UCB cells treated with two LRPPRC shRNAs (Left). qRT-PCR for circANKHD1 in UCB cells transfected with control vector or LRPPRC overexpression plasmid (Right). Data are presented as fold change relative to T24-NC, TCC-SUP-NC or UM-UC-3-Vec cells ±SD of three experiments, **P < 0.01. (C) qRT-PCR for the abundance of circANKHD1 in UCB cells, compared with a non-tumorigenic cell, SV-HUC-1 (Left). Data are presented as fold change relative to SV-HUC-1. LRPPRC is positively associated with circANKHD1 in UCB cells (Right). (D) Verification that circANKHD1 is a circRNA, using divergent and convergent primers. Left, schematic illustration of circANKHD1 locus with specific primers. RT-PCR products with divergent primers showing circularization of circANKHD1. Right, Sanger sequencing to confirm the specific back splicing site of circANKHD1. (E) qRT-PCR for the abundance of circANKHD1 and ANKHD1 mRNA with the treatment of Rnase R in T24 cells. Data are presented as fold change relative to Mock T24 cells ±SD of three experiments, ****P < 0.0001. (F) qRT-PCR for the half-life analysis of circANKHD1 and ANKHD1 mRNAs with the treatment of actinomycin-D in T24 cells. Data are presented as fold change relative to the mRNA expression level at the time point of 0-h ± SD of three experiments, *P < 0.05. (G) Cytoplasmic and nuclear mRNA Fractionation experiment showing that circANKHD1 is mainly located in the cytoplasm. ß-actin and U3 were applied as positive controls in the cytoplasm and nucleus, respectively. Data shown are the means ± SD of three experiments. (H) RNA fluorescence in situ hybridization for circANKHD1. Nuclei were stained with DAPI. Scale bar, 10 µm. (I) A schematic drawing showing the putative binding sites of the miRNAs associated with circANKHD1 and FOXM1. (J) Luciferase reporter assay for the luciferase activity of LUC-circANKHD1 or LUC-circANKHD1-mutant in T24 cells co-transfected with miRNA mimics. Data shown are the means ± SD of three experiments. **P < 0.01. (K) The expression levels of FOXM1 and PRDX3 were enhanced bycircANKHD1 overexpression but were inhibited by miR-671-5p or miR-507 mimics as observed in WB.

Fig 5: LRPPRC regulates cell proliferation and apoptosis in UCB cells. (A) Western blotting reveals that LRPPRC was efficiently knocked down and overexpressed in corresponding cells. (B-C) CCK-8 assays of the cell growth ability of the indicated UCB cells. (D–E) Colony formation assays of the indicated UCB cells. Columns: mean ± standard deviation (SD) of three independent experiments. (F–G) EDU assays of the indicated UCB cells. Columns: mean ± standard deviation (SD) of three independent experiments. (H-I) Apoptosis of UCB cells with modulation of LRPPRC expression was detected by flow cytometric analysis as indicated. Columns: mean ± standard deviation (SD) of three independent experiments. (J) Down expression of LRPPRC in the T24 cell line substantially repressed tumor formation compared with T24-NC cells. Left, images of the xenograft tumors formed in Balb/c nude mice injected with T24-NC or T24-shLRPPRC cells. Right, weights of xenograft tumors (n = 5). **P < 0.01; ***P < 0.001; ****P < 0.0001.