The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

Madeleine Zerbato,Sophie Moniot-Frin,Javier Perea,Anne Galy,Dina Ingrao,Nathalie Holic

doi:10.1371/journal.pone.0058263

Madeleine Zerbato, Sophie Moniot-Frin + Show 4 more

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https://doi.org/10.1371/journal.pone.0058263

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Abstract

Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

Highlights

Prokaryotic and eukaryotic organelle introns are mobile elements able to integrate in the exon junction of an intronless genome [1,2,3]
The Pylaiella littoralis Pl.LSU/2 group II intron is located in the mitochondrial gene encoding the large ribosomal RNA (Fig. 1A; LSU rRNA gene)
This intron contains in its domain IV an openreading frame presenting the predicted conserved domains of group II intron-encoded proteins which are the reverse transcriptase (RT), DNA-binding domain (D), maturase (X) and endonuclease (En) [15,23,25,28,53] (Fig. 1A)

Summary

Introduction

Prokaryotic and eukaryotic organelle introns are mobile elements able to integrate in the exon junction of an intronless genome [1,2,3]. In spite of very divergent mechanisms used for homing, both group I or group II introns rely on the expression of a protein coded by the intron itself (IEP, Intron-Encoded Protein) for the homing process [2] These IEPs often carry different activities: maturase (to help the proper splicing of the intron) [15,16,17,18,19,20,21,22], double strand endonuclease (in group I introns) [17,18,20,22], single strand endonuclease and reverse transcriptase (in group II introns) [23,24,25,26,27,28]. Homing always results in the incorporation of a copy of the intron into the intronless genome

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