Characterizing the host-mitochondria/virus interaction through studies of the L-A dsRNA totivirus.
Viruses and other genetic parasites are present in virtually all forms of life. This chronic condition has led to diverse host cell adaptations such as CRISPR and RNAi, whose functions attenuatethese parasites. We have discovered a role for the mitochondria in viral innate immunity. Yeast are infected with a double stranded RNA (dsRNA) virus called “L-A” that is only vertically transmitted with no extracellular phase. For long thought to be a harmless commensal, we have shown that L-A accumulates to toxic levels in strains lacking the functions of the mitochondrialexoribonucleases Nuc1 and Rex2. Of note, Nuc1 is homologous to endonuclease G, a nuclease that is released from
mitochondria during apoptosis in humans, and some of our studies suggest Nuc1 accumulates in the cytosol in a Por1 dependent manner to accomplish viral attenuation. The L-A RNA dependent RNA polymerase (RdRP) shows homology to those of other RNA viruses such as MERS, SARS, and SARS-Cov-2, the virus that causes the pandemic disease COVID-19. Our ongoing work is focused on the mechanisms of mitochondrial viral attenuation (1) and of RdRP inhibitory drugs such as remdesivir (2). These studies are complimented by further characterization of the L-A
lifecycle using proteomic and genomic approaches to attain a comprehensive cell biological understanding of the host-virus interaction (3).
New tricks for old dogs: novel chromatin targets of histone methyltransferase enzymes.
Histone lysine methylation represents one of the most prominently studied chromatin modifications where they impact transcription and epigenetic inheritance. While the enzymes that control the deposition and removal of lysine methylations on histone residues are evolutionarily ancient and widely conserved, their known substrates are almost exclusively restricted to these solitary lysine residues. This is true even though many of these enzymes have crucial roles in development and disease. We are combining molecular genetic and LC-MS/biochemical approaches to systematically identify new and functionally relevant
We are combining molecular genetic and LC-MS/biochemical approaches to systematically identify new and functionally relevant substrates for numerous conserved histone methyltransferase and demethylases present in yeast. Stemming from this work, we have identified the Nrd1-Nab3-Sen1 (NNS) transcriptional termination complex as a potential enzymatic target of Set1, a conserved histone H3 lysine-4 methyltransferase orthologous to human MLL, and Set3, which is
activated by H3K4me and is itself homologous to MLL5. Specifically, Nab3-K363 is mono-methylated in a manner dependent on Set1 and Set3. Nab3-K363 resides within the Nab3 RNA recognition motif
(RRM) and makes physical contact with nascently transcribed RNA. Our ongoing work is advancing the model that Nab3-K363 undergoes direct methylation by Set1 and/or Set3 and that this methylation impacts Nab3 RNA binding to regulate transcriptional termination in concert with other chromatin modifications. Having established this approach, we are interrogating numerous other chromatin-
associated complexes that participate in the transcription cycle.