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

ABSTRACT: 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 changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.

SUBMITTER: Rajan KS

PROVIDER: S-EPMC10662448 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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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:Trypanosoma brucei and related kinetoplastid parasites possess unique RNA processing pathways, including in their mitochondria, that regulate metabolism and development. Altering RNA composition or conformation through nucleotide modifications is one such pathway, and modifications including pseudouridine regulate RNA fate and function in many organisms. We surveyed pseudouridine synthase (PUS) orthologs in trypanosomatids, with a particular interest in mitochondrial enzymes due to their potential importance for mitochondrial function and metabolism. Trypanosoma brucei mitochondrial (mt)-LAF3 is an ortholog of human and yeast mitochondrial PUS enzymes, and a mitoribosome assembly factor, but structural studies differ in their conclusion as to whether it has PUS catalytic activity. Here, we generated T. brucei cells that are conditionally null (CN) for mt-LAF3 expression and showed that mt-LAF3 loss is lethal and disrupts mitochondrial membrane potential (ΔΨm). Addition of a mutant gamma ATP synthase allele to the CN cells permitted ΔΨm maintenance and cell survival, allowing us to assess primary effects on mitochondrial RNAs. As expected, these studies showed that loss of mt-LAF3 dramatically decreases levels of mitochondrial 12S and 9S rRNAs. Notably, we also observed decreases in mitochondrial mRNA levels, including differential effects on edited vs. pre-edited mRNAs, indicating that mt-LAF3 is required for mitochondrial rRNA and mRNA processing, including of edited transcripts. To assess the importance of PUS catalytic activity in mt-LAF3 we mutated a conserved aspartate that is necessary for catalysis in other PUS enzymes and showed it is not essential for cell growth, or maintenance of ΔΨm and mitochondrial RNA levels. Together, these results indicate that mt-LAF3 is required for normal expression of mitochondrial mRNAs in addition to rRNAs, but that PUS catalytic activity is not required for these functions. Instead, our work, combined with previous structural studies, suggests that T. brucei mt-LAF3 acts as a mitochondrial RNA-stabilizing scaffold.

Project description:RNA-binding proteins (RBPs) are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long unrelated gene arrays. In this report, we present a systematic analysis of an essential RBP, RBP42, in the mammalian-infective bloodstream form of African trypanosome and show that RBP42 is a key regulator of parasite's central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Global quantitative transcriptomic and proteomic analyses reveal that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a positive translational regulatory role of RBP42. Significant changes in central carbon metabolic intermediates, following loss of RBP42, further support its critical role in cellular energy metabolism. Trypanosoma brucei infection, transmitted through the bite of blood-feeding tsetse flies, causes deadly diseases in humans and livestock. This disease, if left untreated, is almost always fatal. Existing therapies are toxic and difficult to administer. During T. brucei's lifecycle in two different host environments, the parasite progresses through distinctive life stages with major morphological and metabolic changes, requiring precise alteration of parasite gene expression program. In the absence of regulated transcription, post-transcriptional processes mediated by RNA-binding proteins play critical roles in T. brucei gene regulation. In this study, we show that the RNA-binding protein RBP42 plays crucial roles in cellular energy metabolic regulation of this important human pathogen. Metabolic dysregulation observed in RBP42 knockdown cells offers a breadth of potential interest to researchers studying parasite biology and can also impact research in general eukaryotic biology.

Dataset Information

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

Publications

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

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