RIOK2 transcriptionally regulates TRiC and dyskerin complexes to prevent telomere shortening

Abstract

Telomere shortening is a prominent hallmark of aging and is emerging as a characteristic feature of Myelodysplastic Syndromes (MDS) and Idiopathic Pulmonary Fibrosis (IPF). Optimal telomerase activity prevents progressive shortening of telomeres that triggers DNA damage responses. However, the upstream regulation of telomerase holoenzyme components remains poorly defined. Here, we identify RIOK2, a master regulator of human blood cell development, as a critical transcription factor for telomere maintenance. Mechanistically, loss of RIOK2 or its DNA-binding/transactivation properties downregulates mRNA expression of both TRiC and dyskerin complex subunits that impairs telomerase activity, thereby causing telomere shortening. We further show that RIOK2 expression is diminished in aged individuals and IPF patients, and it strongly correlates with shortened telomeres in MDS patient-derived bone marrow cells. Importantly, ectopic expression of RIOK2 alleviates telomere shortening in IPF patient-derived primary lung fibroblasts. Hence, increasing RIOK2 levels prevents telomere shortening, thus offering therapeutic strategies for telomere biology disorders.

Fig. 1. Loss of RIOK2 results in telomere shortening.

a Immunoblot showing knockdown (KD) and knockout (KO) of RIOK2 in TF-1 cells using 2 guide RNAs each: KD#1, KD#2 and KO#1, KO#2. 2 different replicates for KD#2 (KD#2.1, 2.2) and KO#2 (KO#2.1, 2.2) are shown. b Proliferation in TF-1 cells after KD and KO of RIOK2 using 2 guide RNAs each: KD#1, KD#2 and KO#1, KO#2; n = 2 independent experiments run simultaneously and plotted together. c Cell cycle analysis of control (Ctrl) vs RIOK2 KD and KO TF-1 cells after days 4, 6, and 8 of gene-editing; n = 3 experimental replicates. d Gene set enrichment analysis (GSEA) plot of telomere maintenance-associated genes from RNA-sequencing in RIOK2-depleted vs control HSPCs. e GSEA plot of telomere maintenance-associated genes from ATAC-sequencing in RIOK2-depleted vs control HSPCs. Chromatin accessibility at the promoters of genes were analyzed and GSEA analyses performed. f, g, h qPCR-based analysis of telomere lengths in HSPCs, TF-1 and K562 cells respectively, upon RIOK2 deficiency. Equal amounts of genomic DNA (gDNA) were loaded across samples to assess telomere lengths/content by measuring T/S ratio (telomere/single copy gene expression), as previously described^,; n = 3 primary human donors in figure f and n = 3 experimental replicates in figures g and h. i, j Fluorescence in-situ hybridization (FISH) of telomeric DNA repeats (TTAGGG) upon RIOK2 deficiency in TF-1 and K562 cells, respectively. Representative images from 3 experimental replicates. Scale bar 10 µm. k, l qPCR-based analysis of telomere lengths in control vs RIOK2-depleted HeLa and HEK293 cells, respectively. n = 3 experimental replicates. m Schematic illustration of assessment performed on MDS patient-derived bone marrow (BM) cells. n Correlation of the mRNA expression of RIOK2 with telomere lengths in MDS patient-derived BM cells; n = 40. AU: arbitrary unit. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test performed in c, f, g, h, k and l. Data represented as mean ± SEM. All comparisons are done w.r.t. control (Ctrl). Two-tailed nonparametric Spearman correlation performed in n and Spearman correlation coefficient (r) and P values are shown. ns: non-significant. See also Supplementary Fig. 1. Source data are provided as a Source Data file.

Fig. 2. RIOK2 transcriptionally regulates TRiC complex expression.

a Volcano plot showing differentially expressed TRiC complex genes (cut off: adj P value < 0.05; log2foldchange-RIOK2 KO/Control) from bulk RNA sequencing of RIOK2 knockout (KO) versus control primary human HSPCs, n = 3 donors. log2foldchange-RIOK2 KO/Control plotted as x-axis and -log10(adj P value) calculated using one-sided ANOVA plotted as Y axis. b Heat map of differentially expressed TRiC complex genes (cut off: adj P value < 0.05) from bulk RNA sequencing of primary human HSPCs with knockdown (KD) and knockout (KO) of RIOK2, n = 3 donors (Don 1,2,3: Donor 1,2,3; Ctrl: Control). Normalized hit counts plotted; one-sided ANOVA. c TCP1 (CCT1)-CCT8 mRNA levels (normalized to β-actin encoded by ACTB) in healthy donor-derived HSPCs upon KD and KO of RIOK2, n = 6 donors. Ctrl: Control. d Chromosome view plots depicting chromatin accessibility at the promoters of TCP1, CCT4, CCT6A and CCT8 in control vs RIOK2 KO HSPCs (ATAC-sequencing). e Relative binding of RIOK2 to the promoter regions of TCP1 and CCT8 via ChIP using monoclonal (mAb) and polyclonal (pAb) antibodies against RIOK2; n = 3 experimental replicates. f Quantification of TCP1 and CCT8-promoter driven luciferase activity in response to dose dependently increasing RIOK2 expression in HEK293 cells; EV: empty vector, WT: wild-type RIOK2. pglev: luciferase reporter plasmid backbone + empty vector; n = 4 and 3 experimental replicates in TCP1 and CCT8 respectively, run parallely. (g, h) Immunoblots and adjoining quantifications showing total protein levels of TCAB1 after KD and KO of RIOK2 in TF-1 and K562 cells, respectively; n = 3 experimental replicates. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test in c, f, g and h, unpaired t-test in e. Data represented as mean ± SEM. All comparisons are done w.r.t. control (Ctrl) in c, g and h, IgG IP in e, and EV in f. See also Supplementary Fig. 2. Source data are provided as a Source Data file.

Fig. 3. RIOK2 regulates mRNA expression of dyskerin complex subunits.

a Differentially expressed dyskerin complex genes from RNA sequencing of RIOK2 KO versus control primary human HSPCs, n = 3 donors. log2foldchange-RIOK2 KO/Control plotted as x-axis and -log10(adj P value) calculated using one-sided ANOVA plotted as Y axis. b Differentially expressed dyskerin genes (cut off: adj P value < 0.05) in HSPCs with KD and KO of RIOK2, n = 3 donors (Don 1,2,3: Donor 1,2,3; Ctrl: Control). Normalized hit counts plotted; one-sided ANOVA. c mRNA levels of DKC1, NHP2, NOP10 and GAR1 (normalized to β-actin encoded by ACTB) in HSPCs upon KD and KO of RIOK2, n = 6 donors. Ctrl: Control. d Chromatin accessibility at the promoters of DKC1, NHP2, NOP10 and GAR1 in control vs RIOK2 KO HSPCs (ATAC-sequencing). e Relative binding of RIOK2 to the promoter regions of DKC1 and NHP2 via ChIP using monoclonal (mAb) and polyclonal (pAb) antibodies against RIOK2; n = 3 experimental replicates. f DKC1 and NHP2-promoter driven luciferase activity with dose dependently increasing RIOK2 expression in HEK293 cells; EV: empty vector, WT: wild-type RIOK2. pglev: luciferase reporter plasmid backbone+empty vector; n = 4 and 3 experimental replicates in DKC1 and NHP2 respectively, run parallelly. g, h Agarose gel picture and quantification showing TERC and 28sRNA levels in control (Ctrl), RIOK2 KD and KO HSPCs. n = 3 donors. i, j TRAP assay showing telomerase activity in TF-1 cells after KD and KO of RIOK2. 0.5–0.25-0.1 µg total protein containing lysates loaded for each condition; n = 3 independent data points derived from 3 different total protein containing lysates in each condition. k, l TRAP assay showing telomerase activity in K562 cells after KD and KO of RIOK2. n = 3 independent data points from 3 different total protein containing lysates in each condition. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test in c, f, h, j and l, unpaired t-test in e. Data represented as mean ± SEM. All comparisons are done w.r.t. control (Ctrl) in c, h, j and l, IgG IP in e, and EV in f. See also Supplementary Figs. 2, 3 and 4. Source data are provided as a Source Data file.

Fig. 4. Loss of RIOK2’s transcriptional functions results in telomere shortening.

a Proliferation in RIOK2 knockout (KO) TF-1 cells ectopically expressing EV (empty vector), WT (wild-type), DBM (DNA-binding mutant), ΔTAD1 or ΔTAD2 (Transactivation domain-deleted mutants) RIOK2. SCR: Scrambled. b Cell cycle analysis of RIOK2 KO TF-1 cells ectopically expressing EV, WT, DBM, ΔTAD1, or ΔTAD2 RIOK2; n = 3 independent experiments run simultaneously and plotted together. c Relative binding of EV, WT, and DBM RIOK2 to the promoters of TCP1 and DKC1 via ChIP using monoclonal antibodies against HA; n = 3 experimental replicates. d Luciferase reporter assay showing transactivation of TCP1 and DKC1 by EV, WT, DBM, ΔTAD1 and ΔTAD2 RIOK2. pglev: luciferase reporter plasmid backbone + empty vector; n = 4 and 3 experimental replicates in TCP1 and DKC1 respectively, run parallelly. e TCP1 and CCT6A mRNA levels (normalized to β-actin encoded by ACTB) in RIOK2 KO TF-1 cells ectopically expressing EV, WT, DBM, ΔTAD1 and ΔTAD2 RIOK2. n = 3 technical replicates- representative quantification shown from n = 3 experimental replicates. f DKC1 and NHP2 mRNA levels (normalized to β-actin encoded by ACTB) in RIOK2 KO TF-1 cells ectopically expressing EV, WT, DBM, ΔTAD1 and ΔTAD2 RIOK2. n = 3 technical replicates- representative quantification shown from n = 3 experimental replicates. g TERC expression (normalized to 28 sRNA) in RIOK2 KO TF-1 cells ectopically expressing EV, WT, DBM, ΔTAD1, ΔTAD2 or K123A RIOK2. n = 3 technical replicates- representative quantification shown from n = 3 experimental replicates. h TRAP assay showing telomerase activity in RIOK2 KO TF-1 cells ectopically expressing EV, WT, DBM, ΔTAD1, ΔTAD2 or K123A RIOK2. i Fluorescence in-situ hybridization of telomeric DNA repeats (TTAGGG) in RIOK2 KO TF-1 cells ectopically expressing EV, WT, DBM, ΔTAD1, ΔTAD2 or K123A RIOK2. Scale bar 10 µm. Representative images from n = 3 experimental replicates. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test in c, d, e, f and g. Data represented as mean ± SEM. Scale bar 10 µm. All comparisons are done w.r.t. EV in c and d, KO + EV in e, f, and g. ns: non-significant. See also Supplementary Figs. 5 and 6. Source data are provided as a Source Data file.

Fig. 5. mRNA expression of RIOK2 is reduced in aging individuals.

a RIOK2 mRNA in PBMCs from n = 30 young individuals (19–30 years) versus n = 146 nonagenarians ( ≥ 90 years); Data represented as mean ± SEM. b, c Correlation of mRNA levels of RIOK2 with TRiC and Dyskerin complex genes in PBMCs from n = 30 young individuals (19–30 years) and n = 146 nonagenarians ( ≥ 90 years), respectively. d Association of RIOK2 mRNA levels in whole blood with corresponding age of individuals in a sub-sample of the SHIP cohort; n = 987. Linear mixed effect model with age as exposure and RIOK2 transcript level as outcome. e RIOK2 mRNA levels in whole blood samples from n = 232 younger ( < 40 years: minimum: −2.89, 1. quantile – 1.5*IQR: −1.50, 1. quantile: −0.38, median: 0.04, 3. quantile: 0.73, 3. quantile + 1.5*IQR: 1.84, maximum: 2.80) versus n = 249 older ( > 60 years: minimum: −3.22, 1. quantile – 1.5*IQR: −1.89, 1. quantile: −0.66, median: −0.0020, 3. quantile: 0.57, 3. quantile + 1.5*IQR: 1.81, maximum: 3.11) SHIP participants. Linear mixed effect model with age as exposure and RIOK2 transcript level as outcome. f Association of RIOK2 mRNA levels with TRiC complex genes in whole blood samples from a sub-sample of the SHIP cohort; n = 987. Linear mixed effect model with RIOK2 transcript level as exposure and the target gene’s mRNA levels as outcome. g Association of RIOK2 mRNA levels with dyskerin complex genes in whole blood samples from a sub-sample of the SHIP cohort; n = 987. Linear mixed effect model with RIOK2 transcript level as exposure and the target gene’s mRNA levels as outcome. Unpaired non-parametric Mann–Whitney t-test in a; Two-tailed Pearson’s correlation performed in b and c, and Pearson’s correlation coefficients (r) and P values are shown. Linear mixed effect models were used in d–g (detailed statistical analyses in Methods section). When multiple array probes map to the same gene, the results of all probes are reported. Effect sizes (β-values) are only stated for significant probes after Bonferroni correction for multiple testing, i.e. when p < 0.05/7 = 0.0071 for the TRiC complex and p < 0.05/4 = 0.013 for the dyskerin complex. See also Supplementary Fig. 6. Source data are provided as a Source Data file.

Fig. 6. Ectopic expression of RIOK2 rescues telomere shortening in IPF patient-derived lung fibroblasts.

a Quantification of RIOK2 mRNA in PBMCs derived from n = 45 healthy individuals versus n = 70 IPF patients. b Quantification of RIOK2 mRNA in lung tissues derived from n = 35 healthy versus n = 49 IPF patients. c Quantification of RIOK2 mRNA in primary lung fibroblasts derived from n = 3 IPF patients versus n = 5 healthy individuals (control). d, e Quantification of mRNA expression of TRiC (TCP1, CCT8) and dyskerin (DKC1, NHP2) complex subunits in lung fibroblasts derived from n = 3 IPF patients versus n = 5 healthy individuals (control), respectively. f TERC expression (normalized to 28sRNA) in lung fibroblasts derived from n = 3 IPF patients versus n = 5 healthy individuals (control). g Quantitative PCR-based analysis of telomere lengths in lung fibroblasts derived from n = 3 IPF patients versus n = 5 healthy individuals (control). h Fluorescence in-situ hybridization (FISH) of telomeric DNA repeats (TTAGGG) in lung fibroblasts derived from n = 3 IPF patients versus n = 5 healthy individuals (control). i Immunoblots showing protein levels of RIOK2 upon ectopic expression of EV or wild-type RIOK2 in lung fibroblasts derived from IPF patients and healthy individuals (control). EV: empty vector. OE: overexpression. j mRNA levels of TCP1 and DKC1 after ectopic expression of EV or RIOK2 in n = 3 IPF lung fibroblasts. k Quantitative PCR-based analysis of telomere lengths in n = 3 IPF lung fibroblasts after ectopic expression of EV or RIOK2. l Fluorescence in-situ hybridization (FISH) of telomeric DNA repeats (TTAGGG) in n = 3 IPF lung fibroblasts after ectopic expression of EV or RIOK2. Representative images from 3 experimental replicates shown. m, n Immunofluorescence imaging and quantification of γH2AX foci in n = 3 IPF lung fibroblasts after ectopic expression of EV or wild-type RIOK2 (WT). n = 33 cells counted for IPF patient#174 EV and WT, n = 26 cells counted for IPF patient#179 EV and WT, n = 27 cells counted for IPF patient#181 EV and WT. Unpaired non-parametric Mann–Whitney t-test in a–g, and n. Paired t-test in j and k. Data represented as mean ± SEM. Scale bar 10 µm. OE: overexpression; See also Supplementary Figs. 7 and 8. Source data are provided as a Source Data file.

Fig. 7. RIOK2 regulates transcription of TRiC and Dyskerin complex subunits in the context of aging, MDS and IPF.

Diagrammatic illustration showing loss of RIOK2-driven transcription of TRiC and dyskerin complexes compromises telomerase activity that triggers telomere shortening in aging individuals, and patients with myelodysplastic syndrome (MDS) and idiopathic pulmonary fibrosis (IPF). Figure 7 was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.