The human long non-coding RNA gene RMRP has pleiotropic effects and regulates cell-cycle progression at G2

Abstract

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.

Figure 1

Transcriptome analysis of fibroblasts from CHH cases and healthy controls. (A) Male individual with CHH with disproportionate short stature and hair hypoplasia. (B) Heatmap showing genes that are significantly differentially expressed between fibroblasts from CHH cases and healthy controls as well as hierarchical clustering of the samples based on the expression of all these genes. Higher expression is marked in orange/red, lower in blue. The gene names (on the left) together with sample codes (above each column) can be visualized in the high-resolution image. An identical list of the genes included in the heatmap, in identical order, together with additional information is available also as Supplementary Table S4. Numbering of patient samples is identical to the numbering in Supplementary Table S1. Cases/controls and passages 1/2/3/4/5 are color-coded. For each of the five cases and five controls, we analyzed 2–4 samples from different passages. (C) Pie chart showing the 25 most enriched Gene Ontology, Biological processes terms (GO_BP categories) in CHH fibroblasts, based on genes that are significantly downregulated in the STRT analysis. Numbers depict the number of genes involved in each category. (D) KEGG Pathway analysis of the genes that are significantly downregulated in CHH fibroblasts (marked in red). Images were obtained by KEGG, Kanehisa Laboratories.

Figure 2

CHH fibroblasts are delayed in G2 phase. (A) Schematic presentation of the setup used in B and C. Fibroblasts were pulse-labeled with EdU and samples were harvested at the indicated time points. (B) CHH fibroblasts are delayed in transition from G2 to G1. The indicated cell lines were followed as outlined in A. Each circle corresponds to one fibroblast. (C) CHH fibroblasts are delayed in transition from G2 to G1. Quantification of fibroblasts denoted in rectangle in B, showing percentage of 4 N cells of EdU positive population 10 h after EdU pulse. Students t-test. Error bars show s.e.m. (D) The graph shows quantification of mitotic duration for the indicated cell lines. There was no major mitotic delay in CHH fibroblasts.

Figure 3

Schematic of key CDK complexes and RMRP function during the cell cycle. CHH fibroblasts delay in G2 phase. Although effects are pleiotropic and affect many cell cycle regulators, the main Cyclin-CDK complexes driving progression through G2 phase show differential regulation in CHH cells. First, mRNA levels of CDK2 that stimulates Cyclin A/B-CDK1 activation at the S/G2 border are reduced. Second, CHH cells show reduced mRNA levels of direct and indirect regulators of CDK1 activity, as Cdc25C and MAST-L.