Poster abstracts
Poster number 92 submitted by Leidy Johana Vanegas Cano
Investigating the role of ribosomal RNA pseudouridylation in regulating translation
Leidy Johana Vanegas-Cano (Biological Chemistry), Rachel O. Niederer (Biological Chemistry)
Abstract:
Pseudouridine (Ψ) is the most ubiquitous modification across eukaryotic ribosomes. Dyskerin (DKC1), the primary pseudouridine synthase responsible for rRNA pseudouridylation, utilizes small nucleolar RNA (snoRNA) as a guide to direct the specific positioning of uridine at its active site, where the enzyme subsequently modifies the rRNA1. In mammalian cells, DKC1 deficiency shows compromised translation initiation and fidelity2,3. Despite evidence linking rRNA pseudouridylation to ribosome function, how these modifications are regulated and their role in selective mRNA translation remains unclear. To investigate this, we examined whether DKC1 expression levels influence ribosome pseudouridylation and mRNA translation. Using the Genotype-Tissue Expression (GTEx) Portal, we selected three cell lines with varying DKC1 gene expression, ranging from low to high abundance: SKOV3 (Ovarian adenocarcinoma), 549 (lung adenocarcinoma), and Hep G2 (hepatocellular carcinoma). Western blot and RT-qPCR analysis showed DKC1 expression differed between cell lines at both the RNA and protein level, which suggests that its regulation depends on the cell type. In vitro and in vivo translation assays revealed significantly higher translation activity in SKOV3 than in A549 and Hep G2, suggesting that intrinsic factors in SKOV3 cells may enhance translation efficiency, possibly through ribosome modifications or associated protein interactions. Additionally, Purified ribosomes from A549 and SKOV3 are active and can restore translation in ribosome-depleted extracts.
References:
(1) Barozzi, C.; Zacchini, F.; Asghar, S.; Montanaro, L. Ribosomal RNA Pseudouridylation: Will Newly Available Methods Finally Define the Contribution of This Modification to Human Ribosome Plasticity? Frontiers in Genetics 2022, 13 (1), 1–7.
(2) Borchardt, E. K.; Martinez, N. M.; Gilbert, W. V. Regulation and Function of RNA Pseudouridylation in Human Cells. Annual Review of Genetics 2020, 54 (1), 309–336. https://doi.org/10.1146/annurev-genet-112618-043830.
(3) Yoon, A.; Peng, G.; Brandenburg, Y.; Zollo, O.; Xu, W.; Rego, E.; Ruggero, D. Impaired Control of IRES-Mediated Translation in X-Linked Dyskeratosis Congenita. Science 2006, 312 (5775), 902–906. https://doi.org/10.1126/science.112383.
Keywords: Ribosome, Translation assay, DKC1