Ribonuclease inhibitor and angiogenin system regulates cell type-specific global translation

Abstract

Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type-specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type-specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non-hematopoietic-origin cells. RNH1-mediated cell type-specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type-specific global translation regulators and highlights the complex translation regulation in vertebrates.

Fig. 1.. Loss of RNH1 decreases translation specifically in hematopoietic-origin cells.

(A and B) Sucrose gradient polysome profiles for wild-type (WT) and corresponding RNH1 knockout (RNH1 KO) nonhematopoietic (A) and hematopoietic (B) origin cells (N = 3). Arrow shows the direction of the sucrose gradient from low to high density. Mean value of absorbance from three independent experiments plotted with the SD (upper panel). Total protein lysates of WT and RNH1 KO cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments (lower panel). (C and D) Schematics of luciferase-expressing plasmid with GAPDH 5′UTR (upper panel). WT and RNH1 KO HeLa and K562 cells were transfected with luciferase-expressing plasmid. Cells were analyzed for Firefly mRNAs by qRT-PCR and normalized to 18S rRNA expression (C) and luciferase protein expression by luciferase assay (D). Data are shown as means ± SEM and are representative of three independent experiments. ***P < 0.001. (E) WT or RNH1 KO K562 and HeLa cells were incubated for 1 hour with OPP, and fluorescence-activated cell sorting (FACS) analysis was performed to measure OPP incorporation. Representative histograms were shown for OPP fluorescence. Data are representative of three independent experiments. (F and G) RNH1 is ectopically expressed in RNH1 KO K562 cells. Cell lysates were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments (F). K562 cells were incubated for 1 hour with AHA, and FACS analysis was performed to measure AHA incorporation. Representative histograms were shown for AHA fluorescence. Data are representative of three independent experiments (G).

Fig. 2.. The absence of RNH1 specifically decreases translation in hematopoietic cells in mice.

(A to F) OPP incorporation assay was performed after 1 hour of intraperitoneal injection with PBS or OPP (50 mg/kg) in WT (Rnh1^fl/fl), hematopoietic-specific Rnh1-deficient (Rnh1^fl/fl, Mx1-Cre⁺), and liver-specific Rnh1-deficient (Rnh1^fl/fl, Alb-Cre⁺) mice. Schematics showing Mx1-Cre model, where Rnh1 was excised by giving three rounds of 200 μg of poly(I:C) using intraperitoneal injections to Rnh1^fl/fl (WT) and Rnh1^fl/flMx1-Cre⁺ (Rnh1^−/−) mice. After 1 week, mice were injected with OPP and organs were harvested after 1 hour (A). Spleen cell lysates of WT and Rnh1^−/− cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments (B). Schematics showing that Rnh1^fl/fl (WT) and Rnh1^fl/fl Alb-Cre⁺ (Rnh1^−/−) mice were injected with OPP, and organs were harvested after 1 hour (C). Liver cell lysates of WT and Rnh1^−/− cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments (D). OPP incorporated spleen (E) and liver (F) tissue sections were stained by CuAAC with TMR-azide and Hoechst (n = 3 mice for each genotype) (scale bar, 100 μm). (G and H) WT or Rnh1^−/− splenocytes, bone marrow cells (G), and hepatocytes (H) were incubated for 1 hour with OPP, and FACS analysis was performed to measure OPP incorporation. Representative histograms were shown for OPP fluorescence (n = 2 mice for each genotype and each mice with three technical replicates).

Fig. 3.. RNH1 is involved in ribosomal protein mRNA translation.

(A) Total protein lysates from WT and RNH1 KO of hematopoietic and non–hematopoietic-origin cell lines were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (B) Schematic showing total and polysomal RNA isolation and RNA-seq analysis. (C and D) The expression of ribosomal and nonribosomal genes in WT and RNH1 KO K562 cells obtained by polysomal RNA-seq (C) or total RNA-seq (D). Total genes with cutoff of P_adj < 0.1 in polysomal RNA-seq and total RNA-seq were selected and plotted. Dotted lines separate genes with log₂(fold change) <1 and >1. (E and F) Plot of log₂(fold change) of total RNA versus translational activity (TA) of ribosomal and nonribosomal genes in WT (E) or RNH1 KO (F) samples. Horizontal dotted lines separate genes with log₂(fold change) <1 and >1. Vertical lines separate genes with arbitrary cutoff of >7 or <7 for log₂(expression in total RNA-seq). (G) The heat maps generated via Metascape tool with default parameters show the relative expression of genes in different gene ontology (GO) gene sets. (H) Protein lysates from K562 and HeLa with WT and RNH1 KO genotypes were analyzed by Western blot for RPS19, RPS3, and RNH1. Blots are representative of three independent experiments. GAPDH is used as a normalizing control, and numbers represent the normalized band intensity with respect to the corresponding WT sample. (I) Protein lysates from WT and Rnh1^−/− bone marrow (BM) and liver were analyzed by Western blot for RPS19, RPS3, and RNH1 (N = 3 mice). GAPDH is used as a normalizing control, and numbers represent the normalized band intensity with respect to the corresponding WT sample.

Fig. 4.. Unaltered mTOR signaling and phosphorylation of eIF2α in the absence of RNH1.

(A) Schematic of mTOR signaling. (B) WT and RNH1 KO HEK293T and K562 cells were treated with or without mTOR inhibitor Torin1 (100 nM) and analyzed by Western blot with the indicated antibodies. Blots are representative of four independent experiments. (C) Schematic of eIF2α kinases mediated translation suppression. (D) WT and RNH1 KO HeLa, HaCaT, HEK293T, K562, Jurkat, and THP1 cell lysates were analyzed by Western blot with the indicated antibodies. Blots presented are representative of two independent experiments. K562 cells treated with sodium arsenate (SA) (100 μM) are used as positive control for stress-induced translation inhibition.

Fig. 5.. RNH1 binds to PABP and helps in mRNA circularization.

(A) Volcano plots depicting RNH1 binding total proteome from WT K562, THP1, and HEK293T cells compared with RNH1 KO of corresponding cells. Plots show the comparisons of log₂(fold changes) (FC) versus adjusted P values (false discovery rate–controlled Benjamini and Hochberg multiple test correction) calculated based on peptide-imputed Top3 (iTop3). (B and C) Whole-cell lysates of WT and RNH1 KO K562 and HeLa cells were used for anti–PABP-IP (B) and anti–eIF4E-IP (C) and then immunoblotted, as indicated. Blots are representative of three independent experiments. (D) Whole-cell lysates of K562 and HEK293T cells were used for m⁷GTP IP and immunoblotting, as indicated. Blots are representative of three independent experiments. (E) Schematics of RNH1-mediated mRNA circularization.

Fig. 6.. RNH1 deficiency increases ANG expression in nonhematopoietic cells.

(A) RNH1 and ANG mRNA expression from single-cell RNA sequencing of different human cells from hematopoietic and nonhematopoietic origin. C0 to C11 are cluster numbers. pTPM, protein-transcripts per million. Data source: https://www.proteinatlas.org. (B) Total protein lysates from hematopoietic- and non–hematopoietic-origin cell lines were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (C) Total protein lysates of different cellular fractions from WT and RNH1 KO of K562 and HeLa cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (D and E) Total protein lysates from WT and RNH1 KO HeLa (D) or HaCaT (E) cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (F) qRT-PCR analysis of WT and RNH1 KO HeLa cells for 47S rRNA, normalized to 18S rRNA. Data are shown as means ± SEM and are representative of six independent experiments. *P < 0.05.

Fig. 7.. ANG compensates for translational defects in RNH1-deficient nonhematopoietic cells.

(A) Total protein lysates from RNH1 KO or RNH1 and ANG double KO HeLa cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (B) RNH1 KO or RNH1 and ANG double KO HeLa cells were incubated for 1 hour with OPP, and FACS analysis was performed to measure OPP incorporation. Representative histograms were shown for OPP fluorescence. Data are representative of three independent experiments. (C) Total protein lysates from WT or ANG KO HeLa cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (D) WT or ANG KO HeLa cells were incubated for 1 hour with OPP, and FACS analysis was performed to measure OPP incorporation. Representative histograms were shown for OPP fluorescence. Data are representative of three independent experiments. (E to G) RNH1 KO K562 cells were transduced with GFP-tagged ANG and incubated for 1 hour with OPP, and FACS analysis was performed to measure OPP incorporation. FACS analysis shows GFP⁺ and GFP⁻ expressing cells. Data are representative of three independent experiments (E). Total protein lysates from control or GFP-tagged ANG-transduced RNH1 KO K562 cells were analyzed by Western blot with the indicated antibodies. Blots are representative of three independent experiments (F). FACS analysis was performed to measure OPP incorporation in ANG-GFP–transduced RNH1 KO K562 cells. Representative histograms were shown for OPP fluorescence (G). Data are representative of three independent experiments. gMFI, geometric mean fluorescence intensity. (H) Illustration of proposed RNH1/ANG–mediated cell type–specific translational regulation.