When a virus infects an animal cell, including a human cell, double-stranded RNA (dsRNA) matching viral sequence is found in the cell. Viral dsRNA is recognized as foreign and an immune response is mounted. Only recently has it been realized that animal cells encode and synthesize their own dsRNA. My laboratory is interested in the poorly understood functions of cellular dsRNA, and further, how cells distinguish the good from the bad— the cellular dsRNA (self) from the viral dsRNA (non-self). To facilitate our studies, we performed genome-wide analyses to map long dsRNA, or Editing Enriched Regions (EERs), expressed in C. elegans, mouse, and human. These "dsRNAomes" show that long dsRNA is predominantly encoded in protein coding genes, in introns (C. elegans and human) or 3' UTRs (mouse). Our current studies use information from dsRNAomes, in vitro molecular biology and biochemistry experiments, and in vivo studies in C. elegans or mammalian cells.
Invertebrates, such as C. elegans and D. melanogaster, use the RNAi pathway to mount an antiviral response. The enzyme Dicer cleaves viral dsRNA to siRNAs, which bind complementary viral transcripts to silence their expression. How does cellular, or "self", dsRNA, escape cleavage by Dicer? Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines in dsRNA. Our studies in C. elegans indicate ADARs mark "self" dsRNA, and this precludes Dicer cleavage. C. elegans strains lacking ADARs, but not wildtype animals, have abundant siRNAs that map to regions of dsRNA revealed in our dsRNAome. Consistent with the idea that the siRNAs mediate silencing, their associated genes are downregulated in animals lacking ADARs. Using CRISPR to precisely remove regions of dsRNA we showed that ADAR regulation of silencing is dependent on long dsRNA. In future studies we will test whether ADAR mutant animals have an aberrant antiviral response, and whether, in addition to their antiviral functions, ADARs regulate silencing of endogenous genes.
Our biochemistry experiments show that Dicer itself can distinguish "self" versus "non-self" dsRNA by recognizing the ends, or termini, of dsRNA. In the absence of ATP, D. melanogaster Dcr-2 (dmDcr-2), cleaves dsRNA substrates with 3' overhanging (3'ovr) termini, which are characteristic of "self" dsRNA, but cannot cleave dsRNA with blunt (BLT) termini, which we speculate mimic termini of viral dsRNA. However, in the presence of ATP, BLT dsRNA becomes the preferred substrate and is cleaved processively. To understand the mechanistic basis for this substrate discrimination, we collaborated with Dr. Peter Shen and used cryo-EM to determine the structure of dmDcr-2 in complex with a BLT dsRNA. These studies indicate that 3'ovr and BLT dsRNA are recognized by different domains of Dicer, something never imagined. Vertebrate Dicer lost the ability to distinguish "self" versus "non-self", and we speculate this occurred as vertebrates evolved to use the interferon pathway, rather than RNAi, to target viral dsRNA. In future studies we will define amino acids that mediate recognition of termini, and differences in these residues in human Dicer. We are interested in understanding how vertebrate and invertebrate systems evolved, and whether we can manipulate the two systems in a controlled manner.
Mouse, human, and C. elegans dsRNAomes. Vertical black lines denote the positions of EERs on the major chromosomes of mouse, human, and C. elegans. Chromosomes are not shown to scale, so horizontal black bars at the bottom display relative chromosome length for each organism.