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

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.

Download Full-text

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011337 ◽

2019 ◽

Vol 294 (51) ◽

pp. 19764-19784 ◽

Cited By ~ 3

Author(s):

Alfred M. Lentzsch ◽

Jun Yao ◽

Rick Russell ◽

Alan M. Lambowitz

Keyword(s):

Reverse Transcriptase ◽

Biological Function ◽

Group Ii Intron ◽

Template Switching ◽

Rna Seq ◽

Switching Mechanism ◽

Group Ii

Download Full-text

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

10.1101/792986 ◽

2019 ◽

Author(s):

Alfred M. Lentzsch ◽

Jun Yao ◽

Rick Russell ◽

Alan M. Lambowitz

Keyword(s):

Nucleic Acids ◽

Group Ii Intron ◽

Template Switching ◽

Reverse Transcriptases ◽

Donor And Acceptor ◽

Nucleotide Addition ◽

Single Base Pair ◽

Group Ii ◽

Template Switch ◽

Template Dna

ABSTRACTThe reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5’ end of one template to the 3’ end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3’-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. 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 non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3’-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3’ end of the acceptor template. The reliance on a single base pair with the 3’ nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3’ end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

Download Full-text

Exon and protein positioning in a pre-catalytic group II intron RNP primed for splicing

Nucleic Acids Research ◽

10.1093/nar/gkaa773 ◽

2020 ◽

Vol 48 (19) ◽

pp. 11185-11198

Author(s):

Nan Liu ◽

Xiaolong Dong ◽

Cuixia Hu ◽

Jianwei Zeng ◽

Jiawei Wang ◽

...

Keyword(s):

Protein Interactions ◽

Active Site ◽

Group Ii Intron ◽

Structural Basis ◽

Structural Rearrangements ◽

Spliceosomal Introns ◽

Structural Impact ◽

Rnp Complex ◽

Group Ii ◽

Structural Insights

Abstract Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the intron-encoded protein (IEP), which is essential for splicing. Although structures of spliced group II intron RNAs and RNP complexes have been characterized, structural insights into the splicing process remain enigmatic due to lack of pre-catalytic structural models. Here, we report two cryo-EM structures of endogenously produced group II intron RNPs trapped in their pre-catalytic state. Comparison of the catalytically activated precursor RNP to its previously reported spliced counterpart allowed identification of key structural rearrangements accompanying splicing, including a remodeled active site and engagement of the exons. Importantly, altered RNA–protein interactions were observed upon splicing among the RNP complexes. Furthermore, analysis of the catalytically inert precursor RNP demonstrated the structural impact of the formation of the active site on RNP architecture. Taken together, our results not only fill a gap in understanding the structural basis of IEP-assisted group II intron splicing, but also provide parallels to evolutionarily related spliceosomal splicing.

Download Full-text