Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

Abstract

Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3'-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3' nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3' end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-retroelement RTs and the RT fingertips loop, with the 3' nucleotide of the acceptor base paired to the 1-nt 3'-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.

Group II Intron-Like Reverse Transcriptases Function in Double-Strand Break Repair by Microhomology-Mediated End Joining

“…Previous findings showed that this activity is dependent upon the RT0 loop, a distinctive conserved structural feature of non-LTR-retroelement RTs, with deletions in the RT0 loop inhibiting the templateswitching activity but not the primer extension activity of both GII and insect R2 element RTs (Jamburuthugoda and Eickbush, 2014; Stamos et al, 2017; Lentzsch et al, 2019). A recent X-ray crystal structure of a template-switching complex of GII RT revealed the structural basis for this activity by showing that the annealing of short base-pairing interactions between the donor and acceptor nucleic acids occurs in a binding pocket that is formed by the RT0 and fingertips loops and is absent in retroviral RTs (Lentzsch et al, 2021).…”

“…Mechanistically, MMEJ and template switching are analogous in requiring the annealing of short microhomologies between two nucleic acid substrates and using the 3' end of one of the annealed strands as a primer for initiating DNA synthesis on the other. A difference, however, is that end-to-end template switching by group II intron RTs is optimal for annealing of a single base pair, while longer base-pairing interactions are inhibitory (Lentzsch et al, 2019), likely reflecting that the 3' ends of the donor and acceptor nucleic acid bind after RT core closure in a tightly constrained binding pocket formed by the RT0 loop and the fingertips loop (Lentzsch et al, 2021). By contrast, the annealing of longer microhomologies, such as those typically used for MMEJ, is more akin to the mechanism used for binding and annealing primers for primer extension, as evidenced by the findings that WT G2L4 and GII A/I RT with I at the active site favors the use of shorter primers and microhomologies, while GII and G2L4 I/A RTs with A at the active site enables more efficient use of longer primers or microhomologies (Figure 3A and C, Figure S10, and Figure S13B and C).…”

“…Surprisingly, we found that a group II intron RT with YADD at its active site could function similarly to G2L4 RT in DNA repair in vitro and in vivo . For both enzymes, a key biochemical activity for DSBR, the ability to anneal short microhomologies between single-stranded 3’-DNA overhangs, was inhibited by mutations in the RT0 loop, a distinctive conserved structural feature of non-LTR-retroelement RTs that is not required for RT activity but was shown previously to function in annealing short microhomologies between donor and acceptor nucleic acids in end-to-end template switching by non-LTR-retroelement RTs (Lentzsch et al, 2019, 2021). These findings and others from the literature discussed below suggest that non-LTR-retroelement RTs may have an inherent ability to function in DSBR in a wide variety of organisms.…”

“…The predicted secondary and tertiary structures of the G2L4 RT closely matched the known structure of GII RT, with the major differences being a longer RT3a insertion and small insertions downstream of RT6 and in the thumb domain (Figure 1B and Figure S3A). The RT0 loop, which plays a critical role in the template-switching activity of group II intron and other non-LTR-retroelement RTs (Jamburuthugoda and Eickbush, 2014;Lentzsch et al, 2019Lentzsch et al, , 2021, is structurally similar between G2L4 and group II intron RTs but differs in having conserved serine residues (Figure S3B). G2L4 RTs also differs from group II intron RTs in lacking C-terminal DNA-binding (D) and DNA endonuclease (En) domains, which function in binding and cleaving DNA target sites during group II intron retrohoming (Figure 1A;San Filippo and Lambowitz, 2002).…”

“…A crystal structure of a full-length group II intron RT (Geobacillus stearothermophilus GsI-IIC RT) in complex with template-primer and incoming dNTP showed that group II intron RTs are similar to retroviral RTs in folding into a hand-like structure with fingers, palm, and thumb forming a cleft that binds the template-primer at the RT active site, but with the NTE/RT0 loop and RT2a insertions contributing to tighter binding pockets for template/primer and incoming dNTP that could contribute to the relatively high fidelity and processivity of these enzymes (Mohr et al, 2013;Stamos et al, 2017) (Figure 1B). The NTE/RT0 loop also plays a key role in a proficient group II intron RT template-switching activity that is dependent upon a short base-pairing interaction between the 3' ends of the donor and acceptor nucleic acids (Mohr et al, 2013;Stamos et al, 2017;Lentzsch et al, 2019Lentzsch et al, , 2021.…”