The Patel Lab research is focused on the following questions: (1) how human mitochondrial DNA is replicated and transcribed, and (2) how innate immune sensors, such as the RIG-I-like family of proteins, recognize RNA viruses to stimulate an interferon response. Our goal is to understand how polymerases and helicases in these systems use ATP hydrolysis and motor activities to improve the efficiency and accuracy of their functions. We use a combination of biochemical, biophysical, structural (cryo-EM), single-molecule fluorescence, and cellular assays to uncover the mechanisms underlying these processes. Additionally, we apply computational methods to develop quantitative models of these pathways.
MITOCHONDRIAL DNA TRANSCRIPTION
We have a long-standing interest in single-subunit RNA polymerases, which are used by phages and mitochondria to transcribe their genomes. After years of studying bacteriophage T7 RNA polymerase, which is now a key tool in RNA research and RNA vaccine development, we have turned our focus to mitochondrial RNA polymerases. These polymerases are homologous to T7 RNA polymerase but remain less studied. We have successfully reconstituted mitochondrial transcription complexes and are working to elucidate their transcription initiation mechanisms using biochemical and cryo-EM approaches. To this end, we have developed quantitative assays to measure each step of the transcription process, including promoter recognition, melting, bending, RNA extension rates, bubble collapse, promoter release, transition to elongation, and nucleotide addition fidelity during elongation. Combining these biochemical insights with structural studies allows us to understand how RNA polymerase and transcription factors coordinate their activities at different promoters. Recent cryo-EM studies have provided snapshots of eight intermediates of yeast mitochondrial RNA polymerase involved in RNA synthesis. This research on the human system will be critical for developing tools and insights to address human diseases related to mitochondrial dysfunction.
MITOCHONDRIAL DNA REPLICATION
Our interest in human mitochondrial DNA replication began when Twinkle was identified as the human mitochondrial helicase, showing homology to the bacteriophage T7 helicase we had studied for many years. It was fascinating to discover that several point mutations in Twinkle linked to mitochondrial diseases were the same as those we had identified as helicase-deficient in our genetic screen with T7 helicase. We dedicated significant effort to reconstitute the mitochondrial replisome with DNA polymerase gamma, Twinkle, and single-stranded binding protein. Twinkle is a hexameric ring-shaped helicase that unwinds DNA to assist the polymerase in replicating the mitochondrial genome. We found that while Twinkle is a relatively poor unwindase on its own, it becomes an efficient motor when coupled with DNA polymerase. Additionally, we discovered many non-replicative functions of Twinkle, including DNA annealing and strand exchange activities, whose roles are not yet fully understood. The balance between Twinkle’s replicative and non-replicative functions may be crucial in dysfunctions associated with inherited mutations in Twinkle.
VIRAL RNA SENSORS
Our research into DNA and RNA helicases led us to study the RIG-I family of helicases, which act as viral RNA sensors. This family includes three proteins, RIG-I, MDA5, and LGP2, that are essential components of the innate immune system and act as first responders to RNA viruses entering the cytoplasm. RIG-I and MDA5 detect a broad range of RNA viruses, stimulating a strong interferon-mediated antiviral response to clear the virus, while LGP2 modulates this response. Given the abundance of RNAs in the cytoplasm, we are interested in understanding how these viral RNA sensors distinguish between self and non-self RNAs and how this process is regulated. Our studies have shown that RIG-I uses its ATPase activity to translocate unidirectionally, rapidly dissociating self RNAs while promoting its oligomerization on viral RNAs. We discovered that an acidic, intrinsically disordered linker within RIG-I is crucial for blocking self-RNA binding, thereby preventing inappropriate interferon responses. To uncover key regulatory steps in this pathway, we are investigating gain- and loss-of-function variants of the RIG-I family receptors associated with dysfunctional virus recognition and autoimmune disorders. We have developed specific assays to interrogate each step in the pathway from RNA recognition to RIG-I activation. These assays are used to screen for RIG-I agonist and antagonists.