This piece was written live during the 5th annual RNA Symposium: Processing RNA. Follow us on Twitter or the tag #umichrna
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Welcome to Dr. Brenda Bass, a senior professor of Biochemistry at the University of Utah. Dr. Bass worked with Dr. Cheque on enzymatic binding sites during her PhD and will be talking to us about self- and non-self detection of mRNA.
When Dr. Brenda Bass began studying long double-stranded RNA (ldsRNA) during her post-doc, it was known that the only time that cells contain dsRNA was after viral infection. After infection, binding proteins attach to viral dsRNA, forming an “SOS”-like signal that initiates an immune response.
Dr. Bass and others had stumbled on some but not all dsRNAs, and thus began compiling the LONG dsRNAome. The self-sequences were found primarily in introns and UTRs (untranslated regions) of protein-coding genes.
Three -omes were considered in the thinking through of the self/non-self identification question – mouse, human, and C. elegans. Later, Drosophila would replace C. elegans as the invertebrate model due to difficulties transitioning from in vivo to in vitro studies of invertebrate dsRNA detection machinery.
Even now, we don’t know the function of many of these ldsRNAs, but we do know that if cells don’t pay attention to these sequences there are consequences for the immune response to viral infection.
Invertebrates use Dicer to recognize and cleave non-self dsRNA before initiating an RNA interference (RNAi) response, whereas vertebrates use a variety of ADAR machinery as part of an interferon response. ADARs are innate immune checkpoints found in both vertebrates and invertebrates, even though these organisms diverged roughly 550 million years ago in evolution.
The central question that this talk will address is exactly how organisms distinguish self from non-self dsRNA.
Termini discrimination is a large piece of this puzzle – invertebrates use Dicer to identify the blunt ends of viral dsRNA and the 3′ end of self RNA. Vertebrate machinery, by contrast, appears to detect the triphosphate end of viral dsRNA and the single, capped phosphate end of self-generated dsRNA.
C. elegans bred without any functional copies of Dicer have misshapen and improperly distributed gonadal cells, but insertion of a Dicer transgene retrieves normal cell morphology and migration. Rescuing transgene in dicer -/- organisms does not, however, rescue endogenous siRNA production.
Dr. Bass’s lab has tried to move from an in vivo to in vitro investigation of Dicer; direct purification of C. elegans Dicer was repeatedly unsuccessful, so the group switched to Drosophila Dicer and went on to purify Dicer protein and produce a Cryo-EM structure.
A cleavage assay for a 106bp dsRNA shows that in the absence of ATP there is no cleavage of blunt dsRNA, whereas dsRNA with 3′ overhangs shows only slight cleavage in the absence of ATP. Conversely, blunt dsRNA is cleaved at significantly higher levels than 3′ overhang dsRNA when ATP is present. Why?
The Bass lab believed that if a 3′ overhang was present it would bind to Helicase domain 2.1 in an ATP-dependent manner, while viral dsRNA would bind the Helicase domain 1 (which is not required for processing of miRNA) to be cleaved independent of ATP. This would provide a neat and efficient way to differentiate endogenous dsRNA from viral dsRNA while also mounting an immune response in a controlled manner.
A combination of Cryo-EM and predictive modeling animation reveal that viral dsRNA is unwound by the two adjacent helicase domains in Dicer through the kinetically favorable process of threading that also allows the dsRNA to pass by binding sites that sit throughout the Dicer complex and cleave the dsRNA if it contains a matching sequence.
The process of unwinding the viral dsRNA prior to cleavage (which requires a reannealed dsRNA strand) appears to be vital to the self/non-self recognition process as an additional checkpoint: perhaps by binding ssRNA (single-stranded RNA), different combinations of these sites within Dicer chemically confirm that the viral dsRNA is in fact of viral origin before initiating an energetically costly immune response.
Dr. Brenda Bass received her Bachelor of Arts in Chemistry from Colorado College, and she went on to get her Ph.D. from the University of Colorado, Boulder in Chemistry. She was a postdoctoral fellow at the Fred Hutchinson Cancer Research Center prior to joining the faculty of the University of Utah School of Medicine. Dr. Bass was welcomed to the National Academy of Science in 2015 for her work investigating double-stranded RNA-mediated pathways.
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