Poster abstracts
Poster number 139 submitted by Leidy Johana Vanegas Cano
Investigating the role of ribosomal RNA pseudouridylation in regulating translation
Leidy Vanegas-Cano (Department of Biological Chemistry, University of Michigan), Rachel O. Niederer (Department of Biological Chemistry, University of Michigan)
Abstract:
Pseudouridine is the most ubiquitous modification across eukaryotic ribosomes. Dyskerin (DKC1), the primary pseudouridine synthase responsible for rRNA pseudouridylation, utilizes small nucleolar RNA as a guide to direct the specific positioning of uridine at its active site, where the enzyme subsequently modifies the rRNA[1,2]. In mammalian cells with DKC1 deficiency also show compromised translation initiation and fidelity[2,3]. Although these studies indicate that rRNA pseudouridylation regulates translation by the ribosome, there is still limited understanding of how these modifications are regulated and their potential effects on selective mRNA translation. To gain insights into how rRNA pseudouridylation regulates ribosome function, we started by investigating whether DKC1 expression levels affect ribosome pseudouridylation and mRNA translation. First, we used the Genotype-Tissue Expression (GTEx) Portal to select three cell lines from tissues with differential expression of DKC1, ranging from low to high abundance (SKOV3, A549, and Hep G2). To validate the differential expression of DKC1 among these three cell lines, we performed western blots monitoring DKC1. Our preliminary results show lower DKC1 expression in SKOV3 compared to A549 and Hep G2, which is in agreement with the expression data from the GTEx Portal. Additionally, to evaluate the overall translation activity of these cell lines, we performed in vitro translation assays using cells extracts derived from each cell line. The results showed that SKOV3 extracts exhibited significantly higher translation activity than Hep G2 and A549 suggesting that high levels of DKC1 might impact translation possibly through ribosome pseudouridylation. Future experiments using direct RNA sequencing using Nanopore will elucidate the rRNA pseudouridylation level in these cell lines as well as any cell-specific Ψ sites. This work constitutes the first step in characterizing the effect of natural variation in DKC1 expression levels on rRNA pseudouridylation in different cell types and will allow us to investigate the impact of rRNA pseudouridylation in mRNA recruitment by the ribosome.
References:
[1]Keszthelyi, T. M.; Tory, K. The Importance of Pseudouridylation: Human Disorders Related to the Fifth Nucleoside. Biologia Futura 2023, 74, 3–15. https://doi.org/10.1007/s42977-023-00158-3.
[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-LinkedDyskeratosis Congenita. Science 2006, 312 (5775), 902–906. https://doi.org/10.1126/science.112383.
Keywords: DKC1, cell lines, in-vitro translation