Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

Abstract

The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5′ end of one template to the 3′ end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI–IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3′-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3′-DNA overhang complementary to the 3′ nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3′ end of cDNAs synthesized from natural templates. Longer 3′-DNA overhangs progressively decreased the template-switching rate, even when complementary to the 3′ end of the acceptor template. The reliance on only a single bp with the 3′ nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3′ end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement–encoded RTs, as well as the optimization of this activity for adapter addition in RNA- and DNA-Seq protocols.