Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Abstract

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3 ′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

RNA structure through multidimensional chemical mapping

“…With currently tested reverse transcriptases, primer extension is inefficient for lengths beyond 1000 nucleotides even on unmodified transcripts; and, after chemical treatment, modified nucleotides either stop the enzyme (in M 2 and MOHCA-seq) or reduce its processivity (under conditions tested for RING-MaP/MaP-2D). Based on these considerations, structure determination of the 1000-nucleotide RNA would appear to be the upper limit for current MCM methods, and even then would require significant methods development, such as characterization of newly available reverse transcriptases (Mohr et al 2013). These calculations indicate that application of MCM to infer structures larger than 1000 nucleotides will require a fundamental advance in the methodology.…”

Enzyme engineering through evolution: Thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology

“…As would be predicted from their biological function, group II intron RTs have higher processivity, fidelity, and thermostability than retroviral RTs (Mohr et al 2013). Retroviral RTs copy their own genomes, or heterologous templates, with high error rates relative to cellular DNA polymerases as an 3 Corresponding authors…”

“…After many years of groundwork, the Lambowitz group reports in RNA a landmark study attaining robust high-level recombinant expression of group II intron RTs, focusing on thermostable enzymes from the bacterial thermophiles Thermosynechococcus elongatus and Geobacillus stearothermophilus (Mohr et al 2013). The methods used to produce these thermostable group II intron RTs are generalizable to the group II intron RTs from other evolutionary branches of the tree of life.As would be predicted from their biological function, group II intron RTs have higher processivity, fidelity, and thermostability than retroviral RTs (Mohr et al 2013). Retroviral RTs copy their own genomes, or heterologous templates, with high error rates relative to cellular DNA polymerases as an 3 Corresponding authors…”

“…Furthermore, due to a physiological dependence on protein and RNA cofolding, RTs other than retroviral family members have not been amenable to recombinant protein expression. After many years of groundwork, the Lambowitz group reports in RNA a landmark study attaining robust high-level recombinant expression of group II intron RTs, focusing on thermostable enzymes from the bacterial thermophiles Thermosynechococcus elongatus and Geobacillus stearothermophilus (Mohr et al 2013). The methods used to produce these thermostable group II intron RTs are generalizable to the group II intron RTs from other evolutionary branches of the tree of life.…”

“…For example, thermostability of the group II intron RTs allows cDNA synthesis at temperatures that denature template RNA secondary structure, and their high processivity enables the synthesis of long cDNAs with minimal background from premature termination (Mohr et al 2013). Perhaps the most striking and unanticipated property of the group II intron RTs is their ability to perform end-to-end template switching.…”