DNA cleavage and reverse splicing of ribonucleoprotein particles reconstituted in vitro with linear RmInt1 RNA

Abstract

ABSTRACTThe RmInt1 group II intron is an efficient self-splicing mobile retroelement that catalyzes its own excision as lariat, linear and circular molecules. In vivo, the RmInt1 lariat and the reverse transcriptase (IEP) it encodes form a ribonucleoprotein particle (RNP) that recognizes the DNA target for site-specific full intron insertion via a two-step reverse splicing reaction. RNPs containing linear group II intron RNA are generally thought to be unable to complete the reverse splicing reaction. Here, we show that reconstituted in vitro RNPs containing linear RmInt1 ΔORF RNA can mediate the cleavage of single-stranded DNA substrates in a very precise manner with the attachment of the intron RNA to the 3´exon as the first step of a reverse splicing reaction. Notably, we also observe molecules in which the 5´exon is linked to the RmInt1 RNA, suggesting the completion of the reverse splicing reaction, albeit rather low and inefficiently. That process depends on DNA target recognition and can be successful completed by RmInt1 RNPs with linear RNA displaying 5´ modifications.