The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway

Ll.LtrB RNA circles form RNPs in vivo. A primer pair was designed to amplify both the lariat (138 bp) and the circular intron junctions (144 bp) from the same RT-PCR reaction (A–C). An alternative RT-PCR strategy was used to amplify complete intron RNA circles (D–E). (A) RT-PCR performed on total RNA extracted from aliquots of a bacterial culture (NZ9800ΔltrB/pDL-P₂₃²-Ll.LtrB-WT) taken at different time points. (B) RT-PCR performed on total RNA extracted from aliquots of bacterial cultures (7 h) expressing different Ll.LtrB variants. The slower migrating bands amplified from the Ll.LtrB-ΔLtrA and Ll.LtrB-LtrAMat⁻ RNA extracts correspond to non-specifc amplifications. (C) RT-PCR performed on intron RNA recovered by immunoprecipitation of the LtrA protein from total cell lysates. (D) Schematic of intron RNA circles showing the position of the two primers used to amplify complete intron RNA circles by RT-PCR. One primer is complementary to the perfect intron circle junction base-pairing with 11 nt on each sides (D0) while the other primer is only 5 nt away (D1). (E) RT-PCR of complete intron RNA circles using intron RNA isolated by immunoprecipitation of the LtrA protein.