title: 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.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Sabine
authors.initials: S
authors.lastname: Mohr
authors.forename: Eman
authors.initials: E
authors.lastname: Ghanem
authors.forename: Whitney
authors.initials: W
authors.lastname: Smith
authors.forename: Dennis
authors.initials: D
authors.lastname: Sheeter
authors.forename: Yidan
authors.initials: Y
authors.lastname: Qin
authors.forename: Olga
authors.initials: O
authors.lastname: King
authors.forename: Damon
authors.initials: D
authors.lastname: Polioudakis
authors.forename: Vishwanath R
authors.initials: VR
authors.lastname: Iyer
authors.forename: Scott
authors.initials: S
authors.lastname: Hunicke-Smith
authors.forename: Sajani
authors.initials: S
authors.lastname: Swamy
authors.forename: Scott
authors.initials: S
authors.lastname: Kuersten
authors.forename: Alan M
authors.initials: AM
authors.lastname: Lambowitz
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015723:Gene Library; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D035683:MicroRNAs; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D012194:RNA-Directed DNA Polymerase
keywords: miRNA; next-generation sequencing; qRT-PCR; retrovirus; transcriptome