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

ABSTRACT: 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.

ORGANISM(S): synthetic construct

PROVIDER: GSE138200 | GEO | 2019/11/11

REPOSITORIES: GEO

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Project description:Pervasive Transcription originates from spurious promoters present in both orientations within coding sequences. Spurious transcripts are typically untranslated and thus for the most part are terminated by transcription termination factor Rho. They can be studied by impairing Rho activity, which in this study was achieved by depleting cells for Rho co-factor NusG. To this end, strains in which the only chromosomal copy of the nusG gene is transcribed from a promoter that can be repressed by arabinose (ARA) were used. This study was aimed at determining how pervasive transcription affect H-NS mediated gene silencing in Salmonella Pathogenicity Island 1 (SPI-1). In particular, the analysis concentrated on the region surrounding the master regulator gene HilD. The activities of promoters in this region were assessed performing semiquantitative 5’RACE-Seq. RNA extracted from cells exposed to the indicated treatments was reverse-transcribed with gene-specific primers in the presence of a template-switching oligonucleotide (TSO). The latter carries three 3’-terminal riboguanosines that hybridize to the polycytosine overhang (typically 3 or 4 Cs) added by the RT enzyme when it reaches the 5’ end of the RNA.This allows the RT enzyme to switch template and polymerise the TSO. Subsequent PCR amplification, with a primer complementary to the TSO and another annealing inside the reverse transcribed region, followed by sequence analysis, allows identifying the 5’ end of the RNA as the sequence lying immediately adjacent to the poly-G track. Performing the PCR step semiquantitatively provides information as to relative RNA levels. Here, the technique was adapted to high throughput sequencing by using PCR primers carrying Illumina adapters at their 5’ ends. Three strains were used, all three carrying the ARA-repressible nusG allele, MA12996, MA14302 and MA14358. This last strain carries a deleted version of SPI-1 with the Anhydrotetracycline (AHTc)-inducible Ptet promoter positioned at edge of the H-NS patch covering hilD, which made it possible to study the effects of NusG depletion in the presence or absence of overlapping transcription.

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

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