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Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes

Removal of the 5′-leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P-medi...

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Autores principales: Ender, Anna, Grafl, Nadine, Kolberg, Tim, Findeiß, Sven, Stadler, Peter F., Mörl, Mario
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8925977/
https://www.ncbi.nlm.nih.gov/pubmed/35022261
http://dx.doi.org/10.1261/rna.078814.121
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author Ender, Anna
Grafl, Nadine
Kolberg, Tim
Findeiß, Sven
Stadler, Peter F.
Mörl, Mario
author_facet Ender, Anna
Grafl, Nadine
Kolberg, Tim
Findeiß, Sven
Stadler, Peter F.
Mörl, Mario
author_sort Ender, Anna
collection PubMed
description Removal of the 5′-leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P-mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5′-leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes—two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5′-leader on substrate recognition by different types of RNase P enzymes.
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spelling pubmed-89259772023-04-01 Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes Ender, Anna Grafl, Nadine Kolberg, Tim Findeiß, Sven Stadler, Peter F. Mörl, Mario RNA Article Removal of the 5′-leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P-mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5′-leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes—two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5′-leader on substrate recognition by different types of RNase P enzymes. Cold Spring Harbor Laboratory Press 2022-04 /pmc/articles/PMC8925977/ /pubmed/35022261 http://dx.doi.org/10.1261/rna.078814.121 Text en © 2022 Ender et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society https://creativecommons.org/licenses/by-nc/4.0/This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) .
spellingShingle Article
Ender, Anna
Grafl, Nadine
Kolberg, Tim
Findeiß, Sven
Stadler, Peter F.
Mörl, Mario
Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes
title Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes
title_full Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes
title_fullStr Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes
title_full_unstemmed Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes
title_short Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes
title_sort synthetic riboswitches for the analysis of trna processing by eukaryotic rnase p enzymes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8925977/
https://www.ncbi.nlm.nih.gov/pubmed/35022261
http://dx.doi.org/10.1261/rna.078814.121
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