Flow Of Gene Expression Across Mitochondrial Condensates

Project Summary/Abstract: Phase transitions are emerging as a fundamental organizational principle for gene regulation. Transcription is organized into diverse higher-order structures called transcriptional condensates: Pol II associates into dynamic foci that rapidly assemble and disassemble concomitant with mRNA production, while the nucleolus maintains a tri-partite layered structure that supports the steady stream of rRNA. However, little is known how and why the flow of gene expression is organized across disparate condensates in the mitochondria. For example, within the mitochondrial matrix, the mitochondrial (mt-) genome and its RNA products are not diffuse but are packaged by proteins into distinct droplet-like structures called mt-nucleoids or mtRNA granules, respectively. Within the framework of biomolecular phase transitions, the vision of the research program will be to investigate how biomolecules involved in mitochondrial gene regulation self- organize into higher-order, functional structures in the mt-matrix and how anomalies to these biophysical processes contribute to mitochondrial (dys)function. The goals for the next five years are to identify the biomolecular interactions underlying the immiscibility of mt-condensates using in vivo and in vitro systems and supported by theoretical modeling. Using super-resolution light microscopy, we will perform biophysical experiments on the organization and dynamics of the components within mt-condensates. We will complement these microscopy experiments with next-generation sequencing approaches to identify the biomolecular networks underlying the biophysical behavior. Next, using our established in vitro mt-transcriptional system, we will study the interplay between phase coexistence and resulting transcriptional activity. We will support these experiments by performing analogous perturbations in live cells within the phase transition framework, allowing us to directly connect phase behavior to the flow of gene expression. Finally, this proposal will elucidate how the structure-function relationships of mt-condensates contribute to mitochondrial (dys)function in live cells. Overall, this research program will apply ideas from thermodynamics and polymer physics to uncover the biophysical principles underlying the flow of gene expression across these disparate condensates within the mt-matrix. This work will shed light on the functional consequences of the canonical organization of mt-condensates and how anomalies contribute to dysfunction, with implications for disease processes. By harnessing mitochondrial biology, we will uncover structure-function relationships of transcriptional condensates, which will pave the way for developing entirely new strategies to precisely target these structures in the pursuit of improving human health.