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Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen

BACKGROUND: Group II introns are RNA enzymes that splice themselves from pre-mRNA transcripts. Most bacterial group II introns harbour an open reading frame (ORF), coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing...

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Autores principales: Ritlop, Christine, Monat, Caroline, Cousineau, Benoit
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410883/
https://www.ncbi.nlm.nih.gov/pubmed/22876289
http://dx.doi.org/10.1371/journal.pone.0041589
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author Ritlop, Christine
Monat, Caroline
Cousineau, Benoit
author_facet Ritlop, Christine
Monat, Caroline
Cousineau, Benoit
author_sort Ritlop, Christine
collection PubMed
description BACKGROUND: Group II introns are RNA enzymes that splice themselves from pre-mRNA transcripts. Most bacterial group II introns harbour an open reading frame (ORF), coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs – U1, U2, U4, U5 and U6) that are important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in trans supports the theory that spliceosomal snRNAs evolved from portions of group II introns. METHODOLOGY/PRINCIPAL FINDINGS: We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation. CONCLUSIONS/SIGNIFICANCE: This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs.
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spelling pubmed-34108832012-08-08 Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen Ritlop, Christine Monat, Caroline Cousineau, Benoit PLoS One Research Article BACKGROUND: Group II introns are RNA enzymes that splice themselves from pre-mRNA transcripts. Most bacterial group II introns harbour an open reading frame (ORF), coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs – U1, U2, U4, U5 and U6) that are important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in trans supports the theory that spliceosomal snRNAs evolved from portions of group II introns. METHODOLOGY/PRINCIPAL FINDINGS: We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation. CONCLUSIONS/SIGNIFICANCE: This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs. Public Library of Science 2012-08-02 /pmc/articles/PMC3410883/ /pubmed/22876289 http://dx.doi.org/10.1371/journal.pone.0041589 Text en © 2012 Ritlop et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ritlop, Christine
Monat, Caroline
Cousineau, Benoit
Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen
title Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen
title_full Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen
title_fullStr Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen
title_full_unstemmed Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen
title_short Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen
title_sort isolation and characterization of functional tripartite group ii introns using a tn5-based genetic screen
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410883/
https://www.ncbi.nlm.nih.gov/pubmed/22876289
http://dx.doi.org/10.1371/journal.pone.0041589
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