PROJECT SUMMARY/ABSTRACT
The proposed research involves continued studies of group II intron and related reverse transcriptases (RTs),
their biological functions, reverse transcription mechanisms, and RNA-seq applications, including the analysis
of human extracellular vesicle and plasma RNAs for RNA diagnostics and liquid biopsy. Group II intron RTs
are encoded by bacterial retroelements called mobile group II introns, which are thought to be evolutionary an-
cestors of introns and retroelements in higher organisms. Group II intron RTs differ structurally and functionally
from retroviral RTs and have novel biochemical properties useful for RNA-seq and other biotechnological appli-
cations. Bacteria also encode a variety of other RTs that are closely related to group II intron RTs, including
RTs that function in RNA spacer acquisition in CRISPR-Cas systems, as well as multiple classes of free-stand-
ing RTs that have acquired cellular functions. At present, little is known about these RTs or their potential for
use in biotechnological applications. In previous work, we developed general methods for expressing group II
intron and related RTs with high yield and activity and applied these methods to group II intron RTs from bacte-
rial thermophiles to obtain Thermostable Group II Intron RTs (TGIRTs). We found that TGIRTs have higher
fidelity and processivity than retroviral RTs as well as a novel template-switching activity that enables facile
RNA-seq adapter addition. Taking advantage of these properties, we developed TGIRT-based methods for
high-throughput RNA sequencing (TGIRT-seq) that enable applications that would be difficult or impossible
with other currently available RTs, including the simultaneous profiling of nearly all RNA biotypes from small
amounts of starting material. We demonstrated the efficacy of these methods for the analysis of human cellu-
lar, extracellular vesicle, and plasma RNAs and have begun to explore their clinical applications. Additionally,
we obtained a first-of-its kind X-ray crystal structure of a full-length TGIRT in complex with template-primer and
incoming dNTP, providing a structural foundation for addressing major questions about the reverse transcrip-
tion mechanism of group II intron RTs, the evolutionary origin of RTs, and the engineering of group II intron
RTs with improved properties for biotechnological applications. In the proposed research, we will investigate
the reverse transcription mechanism and structural basis for the distinctive biochemical properties of group II
intron RTs, explore the evolutionary origin of RTs and their relationship to RNA-dependent RNA polymerases,
continue studies of CRISPR-associated RTs, and extend these studies to new classes of bacterial RTs. Fur-
ther, we will continue to develop TGIRT-seq methods and follow up key findings from our previous TGIRT-seq
analyses of human cellular, extracellular vesicle, and plasma RNAs, including the mechanism and regulation of
extracellular vesicle RNA packaging and secretion, the characterization of novel small non-coding RNAs, and
the functions of short 3’ tRNA fragments. Finally, we will continue to develop clinical applications of TGIRT-seq,
including new approaches for RNA diagnostics and liquid biopsy of cancer and other human diseases.