Isolation and characterization of functional tripartite group II introns using a Tn5-based genetic screen

PLoS One. 2012;7(8):e41589. doi: 10.1371/journal.pone.0041589. Epub 2012 Aug 2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • Conjugation, Genetic
  • DNA Transposable Elements / genetics*
  • Introns*
  • Lactococcus lactis / genetics*
  • Lactococcus lactis / metabolism*
  • Nucleic Acid Conformation
  • RNA Isoforms
  • RNA Splicing
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism
  • Trans-Splicing
  • Transposases / metabolism*

Substances

  • Bacterial Proteins
  • DNA Transposable Elements
  • LtrB protein, Lactococcus lactis
  • RNA Isoforms
  • RNA, Bacterial
  • RNA, Small Nuclear
  • Tn5 transposase
  • Transposases

Grants and funding

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to B.C. B.C. is a FRSQ Chercheur-Boursier Junior 2 and a McGill University William Dawson Scholar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.