Pseudouridylation Of Ribosomal RNA Research Articles

Abstract 2763: A specific ribomethylome pattern in lung cancer is associated with increased risk of metastasis

Abstract Non-coding RNAs drive cancer phenotypes and are associated with patients’ outcome. SnoRNAs are required for the 2’-O-methylation (2’-O-Me) and pseudouridylation of ribosomal RNA (rRNA) thus being essential for ribosomal biogenesis and function. So far, the role of rRNA methylation (ribomethylation) for non-small cell lung cancer (NSCLC) pathogenesis is unknown. To investigate the role of ribomethylation we included samples from 92 patients with lung adenocarcinoma (pathological stage IA to IIB) for comprehensive multi-omics profiling. Amongst transcriptomics, small RNA sequencing, proteomics, and whole exome sequencing, ribomethylation as epitranscriptomic dimension was analyzed using RiboMethSeq. We focused the analysis on complete tumor resection samples. Here 47 out of 106 2’-O-Me sites were fully methylated in all tumor samples suggesting a crucial role for methylation at this position. Surprisingly, 59 sites had a dynamic methylation pattern. By combining ribomethylation sequencing data of dynamic sites with whole-exome-sequencing as well as transcriptome and proteome analyses using a multi-omics factor analysis (MOFA), we discovered a 2’-O-Me signature in a subset of NSCLC patients with a high risk of metastasis and poor prognosis. We termed the patient subset, with underlying ribomethylation signature, epitranscriptomic pro-metastatic phenotype (EPROMET). This phenotype was not associated with genetic mutations as analyzed by exome sequencing. Analysis of gene sets in both transcriptomics and proteomics showed an upregulation of secreted and extracellular matrix proteins in EPROMET patients. Alongside, the ribomethylation site with major contribution to the EPROMET signature; 18S-Um799, mediated through SNORD105/105B, was found to be the most dynamic within the dataset. It also showed the largest difference between EPROMET and non-EPROMET patients. For functional validation, lung cancer cell lines with knockout of snoRNAs contributing to EPROMET signature were generated using CRISPR/Cas9. Lung cancer cells lacking the corresponding rRNA modifications migrated slower in vitro, failed to grow at a distant site in vivo and were impaired in metastasis. Functionally, it was shown that the modulated ribomethylation signature effected translation of secreted proteins by altered mRNA binding to ribosomes. This study indicates the presence of a ribomethylome-associated phenotype of NSCLC related to metastasis and poor prognosis. Altered ribomethylation affects the ribosome function towards differential expression patterns of secreted proteins. Taken together, an epitranscriptomic pattern of 2’-O-Me is associated with invasion/migration properties of NSCLC cells and with development of metastasis. The ribomethylome may present a suitable biomarker and a potential therapeutic target. Citation Format: Cornelius Pauli, Daniel Heid, Christian Rohde, Nadja Krall, Sylvain Delaunay, Michael Kienhoefer, Christian Tischer, Michael Allgäuer, Maximilian Felix Blank, Fengbiao Zhou, Michael Kardorff, Michael Thomas, Hauke Winter, Sarah Sandmann, Marc Kriegsmann, Marc Schneider, Thomas Muley, Alexander Brobeil, Nicole Bäumer, Sebastian Bäumer, Simon Raffel, Albrecht Stenzinger, Peter Schirmacher, Junyan Lu, Judith Zaugg, Michaela Frye, Carsten Müller-Tidow. A specific ribomethylome pattern in lung cancer is associated with increased risk of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2763.

Regulation of translation by ribosomal RNA pseudouridylation.

Pseudouridine is enriched in ribosomal, spliceosomal, transfer, and messenger RNA and thus integral to the central dogma. The chemical basis for how pseudouridine affects the molecular apparatus such as ribosome, however, remains elusive owing to the lack of structures without this natural modification. Here, we studied the translation of a hypopseudouridylated ribosome initiated by the internal ribosome entry site (IRES) elements. We analyzed eight cryo-electron microscopy structures of the ribosome bound with the Taura syndrome virus IRES in multiple functional states. We found widespread loss of pseudouridine-mediated interactions through water and long-range base pairings. In the presence of the translocase, eukaryotic elongation factor 2, and guanosine 5'-triphosphate hydrolysis, the hypopseudouridylated ribosome favors a rare unconducive conformation for decoding that is partially recouped in the ribosome population that remains modified at the P-site uridine. The structural principles learned establish the link between functional defects and modification loss and are likely applicable to other pseudouridine-associated processes.

Ribosomal RNA Pseudouridylation: Will Newly Available Methods Finally Define the Contribution of This Modification to Human Ribosome Plasticity?

In human rRNA, at least 104 specific uridine residues are modified to pseudouridine. Many of these pseudouridylation sites are located within functionally important ribosomal domains and can influence ribosomal functional features. Until recently, available methods failed to reliably quantify the level of modification at each specific rRNA site. Therefore, information obtained so far only partially explained the degree of regulation of pseudouridylation in different physiological and pathological conditions. In this focused review, we provide a summary of the methods that are now available for the study of rRNA pseudouridylation, discussing the perspectives that newly developed approaches are offering.

Small nucleolar RNAs: continuing identification of novel members and increasing diversity of their molecular mechanisms of action.

Identified five decades ago amongst the most abundant cellular RNAs, small nucleolar RNAs (snoRNAs) were initially described as serving as guides for the methylation and pseudouridylation of ribosomal RNA through direct base pairing. In recent years, however, increasingly powerful high-throughput genomic approaches and strategies have led to the discovery of many new members of the family and surprising diversity in snoRNA functionality and mechanisms of action. SnoRNAs are now known to target RNAs of many biotypes for a wider range of modifications, interact with diverse binding partners, compete with other binders for functional interactions, recruit diverse players to targets and affect protein function and accessibility through direct interaction. This mini-review presents the continuing characterization of the snoRNome through the identification of new snoRNA members and the discovery of their mechanisms of action, revealing a highly versatile noncoding family playing central regulatory roles and connecting the main cellular processes.

Early Hematopoietic Progenitors of Dkc1 Hypomorphic Mutant Mice Display Decreased Proliferation Rate and an Impaired Control of Serine/Arginine-Rich Splicing Factor 4 (Srsf4) Translation

The X-linked form of dyskeratosis congenita (X-DC) is caused by mutations in DKC1, which encodes for dyskerin: a putative pseudouridine synthase that mediate the posttranscriptional modification of ribosomal RNA (rRNA) through the conversion of uridine (U) to pseudouridine (Ψ). Patients with X-DC display defects in the pseudouridylation of ribosomal RNA that leads to translational upregulation of IRES-containing mRNAs and affects the affinity of the ribosome for these mRNAs. Studies in vivo and in vitro suggest that the pseudouridylation of ribosomal RNA is the underlying mechanism responsible for the enhanced susceptibility to cancer in these patients. Ruggero et al. have previously reported (Ruggero et al. Science. 2003 Jan 10;299(5604):259-62) that hypomorphic Dkc1m (Dkc1m) mice present pancytopenia associated with hypocellularity of the bone marrow (BM) and increased susceptibility to cancer, therefore constituting a reliable model to study the effect of impaired ribogenesis on hematopoiesis and oncogenesis. However, the cellular and molecular mechanisms leading to BM failure in X-DC remain unknown. Here, we describe the in vivo analysis of the proliferation rate of hematopoietic progenitors in Dkc1m mice and compare the proteomic profile of hematopoietic progenitors between Dkc1m mice and wild-type (WT) controls. For in vivo proliferation assays, 1mg of bromodeoxyuridine (BrdU) was injected intraperitoneally, every 6 hours during 24 hours, in 16 mice (eight WT and eight Dkc1m), and BM cells were harvested by flushing bone cavity, followed by immunofluorescence staining of incorporated BrdU and flow cytometric analysis. No differences were detected in the number of lineage-negative (Lin−), Sca1-positive, c-kit-negative (LSK−) cells, multipotent precursors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP) and immature B (B lin) and erythroid (Eryt) cells between Dkc1m and WT mice. Nevertheless, the BrdU incorporation was lower in LSK cells and CMPs from Dkc1m mice (P<0.05), indicating a lower proliferation rate. Using in vitro stable isotope labelling of amino acids (SILAC) hematopoietic progenitor cells were cultured in complete medium containing 10% fetal bovine serum and cytokines (6 ng/ml mIL-3, 10 ng/ml mIL-6 and 100 ng/ml mSCF). Of note, SILAC is one of the most applied approaches for quantitative proteomics, which uses labeled amino acids contain atoms of different isotopes in cell culture. Briefly, one cell population is cultured in unlabeled medium (control), while a second population is grown in medium substituted with a heavy amino acid (usually arginine 13C and/or lysine 15N). After 2-3 weeks culture, murine hematopoietic progenitors were collected and equal amounts of cells from Dkc1m and WT mice (labeled or not with heavy amino acid) were mixed for protein extraction and analyses. Using this approach, we identified about 3,500 differentially expressed proteins; including proteins related to mRNA assembling and splicing, chromatin remodeling, apoptosis and cell cycle arrest. Interestingly, one of the most differentially expressed proteins between WT and Dkc1m mice (WT light /Dkc1m heavy ratio: 18-fold) was the Serine/arginine-rich splicing factor 4(Srsf4); a member of the splicing factor family (SRSF1, SRSF3 and SRSF4) frequently associated with alternative splicing of genes related to hematopoietic progenitor cell differentiation. Altogether, our preliminary results reveal defects in the transcription/translation of specific mRNAs in Dkc1m cells. Additionally, it is conceivable that the down-regulation of Srsf4 protein could be associated with the low proliferative rate in DKC1m mice and explain the impairment of hematopoiesis in X-DC patients. DisclosuresNo relevant conflicts of interest to declare.

Dyskerin expression influences the level of ribosomal RNA pseudo‐uridylation and telomerase RNA component in human breast cancer

Dyskerin is a nucleolar protein, altered in dyskeratosis congenita, which carries out two separate functions, both fundamental for proliferating cells. One function is the pseudo-uridylation of ribosomal RNA (rRNA) molecules, necessary for their processing, and the other is the stabilization of the telomerase RNA component, necessary for telomerase activity. A significant feature of dyskeratosis congenita is an increased susceptibility to cancer; so far, however, no data have been reported on dyskerin changes in human tumours. Therefore, in this study, the distribution of dyskerin in a large series of human tumours from the lung, breast, and colon, as well as from B-cell lymphomas, was analysed by immunohistochemistry. Dyskerin proved never to be lost or delocalized outside the nucleolus. A quantitative analysis of dyskerin mRNA expression was then performed in 70 breast carcinomas together with the evaluation of telomerase RNA component levels and rRNA pseudo-uridylation. Dyskerin mRNA levels were highly variable and directly associated with both telomerase RNA component levels and rRNA pseudo-uridylation. Dyskerin gene silencing in the MCF-7 human breast carcinoma cell line reduced telomerase activity and rRNA pseudo-uridylation. Significantly, patients with low dyskerin expression were characterized by a better clinical outcome than those with a high dyskerin level. These data indicate that dyskerin is not lost in human cancers and that the levels of its expression and function are associated with tumour progression.

Dyskeratosis congenita and cancer in mice deficient in ribosomal RNA modification.

Mutations in DKC1 cause dyskeratosis congenita (DC), a disease characterized by premature aging and increased tumor susceptibility. The DKC1 protein binds to the box H + ACA small nucleolar RNAs and the RNA component of telomerase. Here we show that hypomorphic Dkc1 mutant (Dkc1m) mice recapitulate in the first and second generations (G1 and G2) the clinical features of DC. Dkc1m cells from G1 and G2 mice were impaired in ribosomal RNA pseudouridylation before the onset of disease. Reductions of telomere length in Dkc1m mice became evident only in later generations. These results suggest that deregulated ribosome function is important in the initiation of DC, whereas telomere shortening may modify and/or exacerbate DC.