A Tn5-based genetic screen to study tripartite class II introns

Ritlop et al. describe the use of a custom-engineered EZ-Tn5™ transposon and a powerful genetic screen to investigate the autocatalytic splicing of group II introns from pre-mRNA. The self-splicing 3.5-kb L1.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was the subject of the study.

The custom transposon contains a pepN transcriptional promoter followed by the P23 L. lactis constitutive promoter. In vitro transposition into a cloned gene created a library of clones with the transposon at every possible position. The genetic screen allowed growth of clones only if the transposon had inserted into the group II intron such that the transcribed RNA fragments were able to assemble, trans-splice, and accurately ligate the two flanking exons. Of 201 independent clones sequenced, the Tn5 transposon was exclusively found in the sense orientation with respect to the intron, such that the terminator and promoter were functional and the intron was transcribed in two fragments. All 201 clones were unique. No clones were identified with the transposon in functionally important regions of the intron.

The study demonstrated the diverse utility of Tn5-based transposons. Further, the discovery that group II introns may be fragmented into two or three pieces and still self-assemble and splice properly provides support for an evolutionary relationship between group II introns and spliceosomal small nuclear RNAs.

Ritlop, C. et al. (2012). Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen PLoS ONE, 7 (8) DOI: 10.1371/journal.pone.0041589