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TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.

ABSTRACT: Following synthesis, RNA can be modified with over 100 chemically distinct modifications, which can potentially regulate RNA expression post-transcriptionally. Pseudouridine (?) was recently established to be widespread and dynamically regulated on yeast mRNA, but less is known about ? presence, regulation, and biogenesis in mammalian mRNA. Here, we sought to characterize the ? landscape on mammalian mRNA, to identify the main ?-synthases (PUSs) catalyzing ? formation, and to understand the factors governing their specificity toward selected targets. We first developed a framework allowing analysis, evaluation, and integration of ? mappings, which we applied to >2.5 billion reads from 30 human samples. These maps, complemented with genetic perturbations, allowed us to uncover TRUB1 and PUS7 as the two key PUSs acting on mammalian mRNA and to computationally model the sequence and structural elements governing the specificity of TRUB1, achieving near-perfect prediction of its substrates (AUC = 0.974). We then validated and extended these maps and the inferred specificity of TRUB1 using massively parallel reporter assays in which we monitored ? levels at thousands of synthetically designed sequence variants comprising either the sequences surrounding pseudouridylation targets or systematically designed mutants perturbing RNA sequence and structure. Our findings provide an extensive and high-quality characterization of the transcriptome-wide distribution of pseudouridine in human and the factors governing it and provide an important resource for the community, paving the path toward functional and mechanistic dissection of this emerging layer of post-transcriptional regulation.

SUBMITTER: Safra M

PROVIDER: S-EPMC5340967 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.

Safra Modi M Nir Ronit R Farouq Daneyal D Vainberg Slutskin Ilya I Schwartz Schraga S

Genome research 20170110 3

Following synthesis, RNA can be modified with over 100 chemically distinct modifications, which can potentially regulate RNA expression post-transcriptionally. Pseudouridine (Ψ) was recently established to be widespread and dynamically regulated on yeast mRNA, but less is known about Ψ presence, regulation, and biogenesis in mammalian mRNA. Here, we sought to characterize the Ψ landscape on mammalian mRNA, to identify the main Ψ-synthases (PUSs) catalyzing Ψ formation, and to understand the fact ...[more]

PMID: 28073919

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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.

Dataset Information

TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.

Publications

TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.

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