Group II Intron and Related Reverse Transcriptases

University Of Texas At Austin, Austin TX

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Abstract

PROJECT SUMMARY/ABSTRACT The proposed research involves continued studies of group II intron and related reverse transcriptases (RTs), their biological functions, biochemical mechanisms, and RNA-seq applications, including analysis of clinical samples for RNA-diagnostics and liquid biopsies. Group II intron RTs are encoded by mobile group II introns, bacterial and eukaryotic organellar retroelements that were the evolutionary ancestors of introns, the RNA splicing apparatus, and retroelements in humans. They differ structurally and functionally from retroviral RTs and have biochemical activities advantageous for RNA-seq and genome engineering applications. Group II intron RTs belong to a larger RT family termed non-LTR-retroelement RTs, which includes human LINE-1 and domesticated bacterial RTs that evolved from group II intron RTs to perform cellular functions. Previously, we developed general methods for purifying group II intron and related RTs with high yield and activity and applied them to group II intron RTs from bacterial thermophiles to obtain Thermostable Group II Intron RTs (TGIRTs). We obtained an X-ray crystal structure of a full-length TGIRT in complex with template-primer and incoming dNTP, a first for any non-LTR-retroelement RT, and used TGIRTs to develop an advantageous high-through- put RNA-seq method (TGIRT-seq) for comprehensive profiling of protein-coding and non-coding RNAs in hu- man cells, extracellular vesicles (EVs), and plasma. During the current grant period, we worked out mecha- nisms used by a related bacterial RT-Cas1 fusion protein to site-specifically integrate RNA spacers into DNA genomes; found that another domesticated bacterial group II-like RT (G2L4 RT) evolved to function in double- strand break repair (DSBR) by microhomology-mediated end joining (MMEJ); used TGIRT-seq to characterize a large novel class of short structured full-length excised linear intron RNAs (FLEXIs) in human cells and plasma; found that FLEXIs and other classes of introns with binding sites for the same subsets of RNA-binding proteins (RBPs) were enriched in functionally related host genes, whose expression might thus be coordinately regulated by these RBPs; developed TGIRT-seq methods for parallel analysis of transcriptional and post-tran- scriptional gene regulation; and used these methods to analyze inflammatory breast cancer (IBC) clinical sam- ples, leading to new insights into IBC and the identification of potential IBC biomarkers in cells and plasma. In the proposed research, we will investigate: (i) the structural basis and physiological significance of novel mech- anisms used for initiation of cDNA synthesis by an RT-Cas1 protein shared by bacterial group II intron and hu- man LINE-1 RTs; (ii) the mechanism and structural basis for DSBR by G2L4 RT, extending these studies to group II intron and LINE-1 RTs for which we found DSBR via MMEJ is an inherent activity; (iii) explore the in- volvement of LINE-1 elements in DSBR in human cells; (iv) use TGIRT-seq to analyze cancers; and (v) investi- gate the physiological significance and mechanisms that lead to enrichment of FLEXIs and intron RNA frag- ments in EVs and plasma, including a class of FLEXIs that may function in gene regulation.