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The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue.

ABSTRACT: The nearly conserved U54 of tRNA is mostly converted to a version of ribothymidine (T) in Bacteria and eukaryotes and to a version of pseudouridine (?) in Archaea. Conserved U55 is nearly always modified to ?55 in all organisms. Orthologs of TrmA and TruB that produce T54 and ?55, respectively, in Bacteria and eukaryotes are absent in Archaea. Pus10 produces both ?54 and ?55 in Archaea. Pus10 orthologs are found in nearly all sequenced archaeal and most eukaryal genomes, but not in yeast and bacteria. This coincides with the presence of ?54 in most archaeal tRNAs and some animal tRNAs, but its absence from yeast and bacteria. Moreover, ?54 is found in several tRNAs that function as primers for retroviral DNA synthesis. Previously, no eukaryotic tRNA ?54 synthase had been identified. We show here that human Pus10 can produce ?54 in select tRNAs, including tRNALys3, the primer for HIV reverse transcriptase. This synthase activity of Pus10 is restricted to the cytoplasm and is distinct from nuclear Pus10, which is known to be involved in apoptosis. The sequence GUUCAm1AAUC (m1A is 1-methyladenosine) at position 53-61 of tRNA along with a stable acceptor stem results in maximum ?54 synthase activity. This recognition sequence is unique for a ? synthase in that it contains another modification. In addition to ?54, SF9 cells-derived recombinant human Pus10 can also generate ?55, even in tRNAs that do not contain the ?54 synthase recognition sequence. This activity may be redundant with that of TruB.

SUBMITTER: Deogharia M

PROVIDER: S-EPMC6380271 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue.

Deogharia Manisha M Mukhopadhyay Shaoni S Joardar Archi A Gupta Ramesh R

RNA (New York, N.Y.) 20181207 3

The nearly conserved U54 of tRNA is mostly converted to a version of ribothymidine (T) in Bacteria and eukaryotes and to a version of pseudouridine (Ψ) in Archaea. Conserved U55 is nearly always modified to Ψ55 in all organisms. Orthologs of TrmA and TruB that produce T54 and Ψ55, respectively, in Bacteria and eukaryotes are absent in Archaea. Pus10 produces both Ψ54 and Ψ55 in Archaea. Pus10 orthologs are found in nearly all sequenced archaeal and most eukaryal genomes, but not in yeast and bac ...[more]

PMID: 30530625

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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

The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue.

Publications

The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue.

Similar Datasets

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