Dataset Information

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

ACCESS DATA

Similar Datasets

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.

Project description:Genomic rearrangements may cause both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements have been proposed such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR). However, to what extent these mechanisms contribute to gene-specific pathogenic copy-number changes (CNCs) remains understudied. Furthermore, only few studies resolved these pathogenic alterations at nucleotide-level resolution. Accordingly, our aim is to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array Comparative Genomic Hybridization (aCGH), we have identified the breakpoints and characterized the likely rearrangement mechanism of the NF1 intragenic CNCs in 78 unrelated patients. Unlike the most typical recurrent rearrangements mediated by flanking low copy repeats (LCRs), NF1 intragenic CNCs have diverse breakpoint locations, and are characterized by different rearrangement mechanisms. We propose the DNA replication-based mechanisms comprising FoSTeS/MMBIR and serial replication stalling to be the predominant mechanism leading to NF1 intragenic CNCs. In addition to the loop of a 197-bp palindrome located in intron 40, four Alu elements located in intron 1, 2, 3 and 50 were also identified as significant intragenic rearrangement hotspots within the NF1 gene. However, no clear genotype-phenotype correlations could be identified among the NF1 patients carrying NF1 intragenic CNCs. Patient DNA samples with non-overlapping CNCs, as estimated by MLPA, were labeled with Cy3 and Cy5 fluorophores respectively, and hybridized onto the microarray. Alternatively, patient DNA was hybridized versus unrelated individual blood DNA. No hybridizations using biological replicates were performed. In total 6 samples were included. Please note that our experimental setup included a hybridization in which two DNA samples with non-overlapping deletions, from two NF1 patients, were hybridized in one experiment (sample codes: 1253 and 1403). In this specific case, assigning test or reference function to the samples was a matter of arbitrary choice. However, if needed, sample 1253 can be denoted as test, and sample 1403 can be denoted as reference in this experiment.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure