Dataset Information

Role of H/ACA snoRNA in translation

ABSTRACT: Trypanosomes are unicellular parasites that cycle between the insect and mammalian host and is the causative agent of African sleeping sickness and the wasting disease, nagana, in cattle. Previous studies from our lab suggested that individual rRNA modification such as pseudouridine (Y) and 2â€™-O-methylation are developmentally regulated during the complex life cycle of these parasites. In this study we identified that the Y composition of translating ribosomes are different from rRNA derived from total RNA. Ablation of single snoRNA guiding Y in one of the inter-subunit bridges of the ribosome affects its affinity to translate specific mRNA. Mutational analysis in the pseudouridylation pocket of this snoRNA supports for the notion that the guided modification and not the chaperonic activity, is essential for the observed phenotype. Structural probing of rRNA using DMS-seq suggested that a single modification can affect the rRNA structure locally as wells as globally. Quantitative profiling of the composition of mutant ribosomes suggested the absence of eS12 ribosomal protein in the mature ribosomes. Addback experiments validated the role of eS12 during the loss of a single Y in these mutant cells. Orthologous experiments in related leishmania spp. also verify importance of a single Y in the function of ribosome. Ongoing experiments intend to visualize such ribosomes lacking a single RNA modification and decipher their significance towards ribosome structure.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Tirza Doniger

LAB HEAD: Prof. Shulamit Michaeli

PROVIDER: PXD030544 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

Rajan K Shanmugha KS Madmoni Hava H Bashan Anat A Taoka Masato M Aryal Saurav S Nobe Yuko Y Doniger Tirza T Galili Kostin Beathrice B Blumberg Amit A Cohen-Chalamish Smadar S Schwartz Schraga S Rivalta Andre A Zimmerman Ella E Unger Ron R Isobe Toshiaki T Yonath Ada A Michaeli Shulamit S

Nature communications 20231120 1

Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These ...[more]

PMID: 37985661

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Project description:Osteoarthritis (OA) is a chronic debilitating joint disease which is strongly associated with ageing. OA involves pathological cellular processes in all joint structures and affects articular cartilage integrity, leading to dysfunctional joint articulation. The biomolecular processes that catalyze the disturbances in the articular chondrocyte phenotype leading to OA are poorly understood, and it is expected that a comprehensive understanding of the avenues leading to catabolic changes and disruption of articular chondrocyte homeostasis will provide important cues for future treatments of the condition. Chondrocytes are specialized secretory cells with highly active protein translational machinery, enabling the synthesis and maintenance of the protein-rich cartilage extracellular matrix (ECM). Disturbances in chondrocyte protein translation in cartilage development and OA are connected to mTOR activity, ER stress, unfolded protein response (UPR)and CHOP-mediated apoptosis. These responses change the downstream translational activity of the biosynthesized ribosome. The assembled mammalian ribosome is built from ribosomal RNAs (rRNAs), together with more than 80 different protein subunits. At the heart of the ribosome, the 18S rRNA guides the decoding of the mRNA message, while an ancient ribozyme activity in the 28S rRNA forms the core of the peptidyltransferase center that polymerizes the amino acid sequence encoded by the mRNA into functional proteins. Post-transcriptional maturation of rRNAs is an integral part of the biosynthesis of ribosomes and ribonucleolytic processing of the major 47S rRNA precursor into mature 18S, 5.8S, and 28S rRNAs is rate limiting for ribosome biogenesis. The U3 small nucleolar RNA (snoRNA) is an evolutionarily highly conserved box C/D-class snoRNA which catalyzes the endoribonucleolytic processing of the 5’ external transcribed spacer (ETS) of the 47S pre-rRNA by base complementarity-guided pre-rRNA substrate recognition and plays a crucial role in the maturation of 18S rRNA. Although extensively studied in yeast, it was only recently demonstrated that U3 snoRNA is indispensable for rRNA maturation in human cells. Pathways controlling ribosome activity have previously been described in the regulation of chondrocyte homeostasis. We here now postulate that not only ribosome activity is involved in chondrocyte homeostasis, but that OA pathophysiological situations can also cause alterations in chondrocyte ribosome biogenesis with consequences for cellular protein translation. Since U3 snoRNA-driven rRNA production is rate-limiting in ribosome biogenesis, we hypothesized that the U3 snoRNA is critical for chondrocyte homeostasis. In this study we therefor aimed to determine whether OA pathophysiological conditions interact with chondrocyte U3 snoRNA levels, thereby influencing rRNA levels and chondrocyte translation capacity.

Dataset Information

Role of H/ACA snoRNA in translation

Publications

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

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