Elucidating how long 3'UTRs form and cause autoinflammation

PROJECT SUMMARY The highly dynamic nature of RNA structure is key to diversifying RNA function. Notably, certain RNA structures can act as immunostimulatory molecules. Our immune system uses various pattern recognition receptors (PRRs) to detect immunostimulatory RNA molecules and trigger inflammation. PRRs are present in all immune and non- immune cells ensuring a rapid and broad immune response. Many PRRs are specialized in detecting long double-stranded RNA (dsRNA) structures, classically thought to be present in the viral genome. Interestingly, our studies, as well as the studies of others, have demonstrated that aberrant sensing of endogenous (self) dsRNAs by PRRs can cause severe autoinflammatory diseases. We recently uncovered that neurons are intrinsically enriched for long dsRNAs. In homeostasis, neuronal dsRNAs stimulated PRRs to produce low (‘tonic’) levels of type I interferon (IFN), which protected neurons from viral infection. However, when dsRNA levels were dysregulated (‘too high’), dsRNAs caused pathological inflammation. Therefore, neuronal dsRNA levels must be tightly regulated within a ‘Goldilocks zone’ to prevent neuroinflammation. Furthermore, our findings suggest that neuronal dsRNAs could be an attractive therapeutic target to control inflammation. However, the identities of neuronal dsRNAs remain enigmatic. We recently identified three genes that induce dsRNAs in neurons. We demonstrated that neuron-enriched genes ELAVL2, ELAVL3, and ELAVL4 (HuB, HuC, and HuD) can increase (i) the length of 3’ untranslated regions (UTRs), (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs (e.g., MDA5, PKR, and TLR3). This finding indicates that dsRNA levels correlate with 3’UTR length, giving rise to the idea that 3’UTRs could be major sites for dsRNA formation in neurons. Indeed, neurons are well known to express the longest average length 3’UTR in the human body. The central hypothesis to be tested in this application is that long 3′UTRs serve as major sources for self-dsRNAs that activate PRRs in neurons. First, we will identify additional ‘genes’ that can lengthen 3’UTRs and give rise to dsRNAs (Aim 1), then we will determine the ‘mechanism’ of how Hu proteins lengthen 3’UTRs and increase dsRNAs (Aim 2). Lastly, we will determine the ‘functional significance’ of 3’UTRs in a disease model for Aicardi–Goutières syndrome (AGS) (Aim 3). AGS is a severe neuroinflammatory disorder that can be caused by aberrant sensing of self-dsRNAs. With the proposed aims, we will test an innovative idea that globally modulating 3’UTR length can fine-tune innate immune responses in cells. Since 3’UTRs and dsRNAs are extremely divergent across species (even between mice and humans), we will utilize various human cells (including human stem cell derived neurons), and apply cutting edge genome engineering, dsRNA imaging, biochemistry, and novel dsRNA sequencing technologies our lab developed. This is a highly collaborative project at the intersection of immunology, RNA biology, and neurobiology. We envision that these studies can lead to novel therapeutic strategies that target RNA or RNA- associated pathways to control inflammation in neural and autoimmune disorders.