A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing

Abstract

The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5'-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5'-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5'-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.

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

“…RT-1 to RT-7 are conserved RT sequence blocks found in all RTs. D denotes the C-terminal DNA-binding domain that functions in recognition of DNA target sites during retrohoming (38). B, outline of template-switching experiments.…”

“…Finally, mobile group II introns, the type of non-LTR retroelement whose RT is studied here, are bacterial and organellar retrotransposons that insert site-specifically into DNA target sites by a process called retrohoming. In this process, the excised intron lariat RNA integrates directly into a DNA strand by reverse splicing and is reverse-transcribed by the RT using either an opposite strand nick or a nascent DNA strand as a primer for reverse transcription (20,38). Group II intron RTs have an end-to-end template-switching activity similar to that of MRP and R2 element RTs (6,39), and this activity has been utilized for adapter addition in RNA-Seq using a thermostable group II intron RT (TGIRT-seq) (6,8,9).…”

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

“…GsI-IIC RT (TGIRT-III) has been used for a variety of applications, including comprehensive profiling of whole-cell, exosomal and plasma RNAs Qin et al 2016;Shurtleff et al 2017;Boivin et al 2018); quantitative tRNA-seq based on the ability of the TGIRT enzyme to obtain full-length end-to-end reads of tRNAs with or without demethylase treatment (Shen et al 2015;Zheng et al 2015;Qin et al 2016); determination of tRNA aminoacylation levels (Evans et al 2017); high-throughput mapping of posttranscriptional modifications by distinctive patterns of misincorporation (Katibah et al 2014;Zheng et al 2015;Shen et al 2015;Shurtleff et al 2017;Li et al 2017;Safra et al 2017); identification of protein-bound RNAs by RIP-Seq or CLIP (Katibah et al 2014;Zarnegar et al 2016); and RNA-structure mapping by DMS-MaPseq (Zubradt et al 2017;Wang et al 2018) or SHAPE (Mohr et al 2018). A study comparing TGIRT-seq to benchmark TruSeq v3 datasets of rRNA depleted (ribodepleted) fragmented Universal Human Reference (UHR) RNA with External RNA Control Consortium (ERCC) spike-ins showed that TGIRT-seq: (i) better recapitulates the relative abundance of mRNAs and ERCC spike-ins; (ii) is more strand-specific;…”

Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools

“…Because of the high processivity of TGIRTs, the resulting RNA-seq data sets showed dramatically more uniform 5 ′ -to-3 ′ read coverage than those obtained in parallel with SuperScript III RT, which over-represented reads near the 3 ′ end of the mRNAs (Mohr et al 2013b). Other applications in which TGIRTs have been used for reverse transcription from an annealed DNA primer include (1) synthesis of long ssDNAs from RNA templates for CRISPR genome editing (Li et al 2017a); (2) reverse transcription and quantitation of GC-rich repeat expansions, which are clinically important in diseases such as myotonic dystrophy and some forms of amyotrophic lateral sclerosis (ALS) (Carrell et al 2017); (3) RNA-structure mapping by DMS modification and SHAPE (Wu and Bartel 2017;Zubradt et al 2017;Mohr et al 2018); and (4) IR-CLIP for the identification of protein-bound RNAs, in which TGIRT gave four-to eightfold higher cDNA yields than SuperScript III (Zarnegar et al 2016). TGIRT has also been used for in vitro Stamos et al 2017.…”