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

Abstract

Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their premRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.

Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria

“…We observed a third productive splicing pathway in the case of trans-splicing nad5 intron 2, one that is analogous to the cis-splicing group II intron pathway which generates a full-length in vivo circularized intron (Murray et al, 2001;Monat et al, 2015). However in the present case, there is a short stretch of non-encoded nucleotides at the junction between the intron 5′ and 3′ ends, and to our knowledge this is the first such report for a trans-splicing intron.…”

Circular RNAs: Regulators of Cancer-Related Signaling Pathways and Potential Diagnostic Biomarkers for Human Cancers

“…CircRNAs are produced by circularization of a single exon 9 , 10 , several exons 11 , exons and introns named exon-intron circRNA (ElcirRNA) 12 , or intronic sequences alone (circularized intron RNA; ciRNA) 13 , 14 . CircRNAs were first found in some plant viroids as covalently closed circular RNA molecules with the base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules 15 .…”

Circularization pathway of a bacterial group II intron

“…Following the initial description of the branching ( 14 – 16 ) and the hydrolysis pathways ( 17 , 18 ), circularization of group II introns was mostly overlooked. Nevertheless, since that time, group II intron RNA circles from various systems were described both in vivo ( 19 – 23 ) and in vitro ( 11 , 24 ). We recently demonstrated that a significant proportion of the model group II intron, Ll.LtrB, from the Gram-positive bacterium Lactococcus lactis , excises as circles in vivo ( 19 ).…”

“…Nevertheless, since that time, group II intron RNA circles from various systems were described both in vivo ( 19 – 23 ) and in vitro ( 11 , 24 ). We recently demonstrated that a significant proportion of the model group II intron, Ll.LtrB, from the Gram-positive bacterium Lactococcus lactis , excises as circles in vivo ( 19 ). Interestingly, we detected intron circles for all the Ll.LtrB mutants studied and demonstrated that both branching and circularization take place concurrently in vivo .…”