UC Berkeley

Mitochondria are essential organelles responsible for a wide array of cellular functions including fatty acid oxidation, immune signaling, and most importantly cellular respiration. They maintain many copies of their own unique genomes which encode for essential electron transport chain proteins, as well as ribosomal and transfer RNAs required for gene expression. Transcription of the mitochondrial genome initiates bidirectionally and produces two polycistronic transcripts that then must undergo processing and maturation in discrete structures adjacent to the mitochondrial DNA called RNA granules. Mitochondrial transcription is initiated heterogeneously and asynchronously, meaning mitochondrial gene expression is regulated by fine-tuned post transcriptional processing and degradation machinery encoded in the nuclear genome. Unlike nuclear gene expression, all steps of the mitochondrial central dogma happen within the confines of the mitochondrial matrix, making it unclear how mature mitochondrial RNAs and their translation machinery are spatially organized across dynamic mitochondrial networks that are constantly undergoing fission and fusion. In the first part of this work, we report that processed mitochondrial RNAs are consolidated into translational hubs distal to either mitochondrial DNA or RNA processing granules in human cells. We found that the highly conserved helicase SUV3 contributes to the distribution of processed RNA within mitochondrial networks, and that perturbations in this pathway lead to an accumulation of dsRNA and a reorganization of mature transcripts into translationally-repressed mesoscale bodies. This reorganization was found to be downstream of dsRNA accumulation and in part dependent on mitochondrial translation. This work reveals that, just as mitochondrial transcription is regulated at nucleoids and RNA processing is regulated at RNA granules, translation by mitoribosomes occurs within defined RNA domains that are dynamically remodeled for quality control.

Similarly to mitochondrial gene expression, the organization of nuclear gene expression machinery into cytoplasmic RNA granules serves to regulate both the where and when of cytoplasmic translation. These RNA and protein rich bodies contain all the necessary components needed for protein production and are remodeled during cellular stress into translationally suppressed stress granules. While cytoplasmic RNA granules serve as important regulatory hubs, allowing for rapid and local responses to changing cellular needs, they also serve as an attractive site for viral replication due to the concentration of gene expression machinery. While work has been studying how viruses may co-opt the cytoplasmic granules for viral replication, relatively little is known about how viruses may adapt replication or infection strategies to exploit the gene expression systems within the mitochondria. Given our new understanding that mitochondria also organize their gene expression systems into translation hubs that are remodeled during stress, we wondered if there was any evidence of viral species exploiting them for their replication. In the second part of this work, we searched thousands of publicly available RNA sequencing runs for novel viral species and report the discovery 763 new viral sequences belonging to the family Mitoviridae, a family of (+)ssRNA viruses that have previously been suggested to interact with mitochondrial gene expression machinery. The identified sequences fill in existing gaps in known mitovirus diversity, and allowed us to further expand the virus family, including previously uncharacterized clades and classes. Using this expanded diversity, we were able to annotate new mitovirus specific protein motifs, and identify hallmarks of mitochondrial translation such as mitochondrial specific codons and codon usage. This work expands the known diversity of mitochondrial viruses, and provides strong evidence for their infection, and use of mitochondria for viral replication.

Together, this work provides novel insights into the regulation of mitochondrial gene expression and how it is reorganized in response to transcriptional stress. It then investigates a family of (+)ssRNA viruses termed Mitoviridae, and provides evidence of their direct co-opting of mitochondrial gene expression machinery for their replication and life cycle. This work provides the foundation to not only explore the functional consequences of the spatial regulation of mitochondrial gene expression, but also to provide evidence of a viral species that may co-opt these translation hubs for their own replication