Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA575026 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10206027.fastq.gz | Fastqsanger.gz | |||
| SRR10206028.fastq.gz | Fastqsanger.gz | |||
| SRR10206029.fastq.gz | Fastqsanger.gz | |||
| SRR10206030.fastq.gz | Fastqsanger.gz | |||
| SRR10206031.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 11 |
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Project description:The 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) 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. Although dependent upon only a single base pair between the donor and acceptor, template switching discriminates against mismatches, which coupled with the high processivity of the enzyme, enables the synthesis of full-length DNA copies of acceptor 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.
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Project description:Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template-DNA primer heteroduplex with a 1-nt 3'-DNA overhang. The structure showed that the 3' end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non-long terminal repeat-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 residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non-long terminal repeat-retroelement RTs and viral RNA-dependent RNA polymerases.
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Project description:Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo. It remains unknown whether similar mechanisms of defense are employed by other related DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.
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