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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 , the efficiency of RNase P-mediated cleava...

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Bibliographic Details
Published in:RNA (Cambridge) 2022-04, Vol.28 (4), p.551-567
Main Authors: Ender, Anna, Grafl, Nadine, Kolberg, Tim, Findeiß, Sven, Stadler, Peter F, Mörl, Mario
Format: Article
Language:English
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Summary: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 , 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 and the ribonucleoprotein of 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.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.078814.121