Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation

Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, -3, -4) can increase 1) 3'UTR length, 2) dsRNA load, and 3) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense ‘self’ dsRNAs to pre-emptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation. For the HuB/C/D ectopic expression experiment (GSE214943; biological triplicates of HEK-293T_Mock, HEK-293T_EV, and HEK-293T_HuB/C/D), RNA was extracted with the Direct-zol RNA MiniPrep (Zymo Research). ERCC RNA Spike-In Mix (Thermo Fisher Scientific) was added to each RNA sample. rRNA was depleted using the RiboMinus™ Eukaryote Kit (Thermo Fisher Scientific). Libraries were prepared with the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs). The 9 libraries were pooled and sequenced on the Illumina Novaseq platform with a read length of 150 nt in the paired end configuration. For analyzing global 3`UTR extension in neurons (GSE214941), biological triplicates of RNA samples from neurons and HEK 293T were extracted with the Direct-zol RNA MiniPrep (Zymo Research). After adding ERCC RNA Spike-In Mix (Thermo Fisher Scientific), libraries were prepared with the TruSeq stranded Total RNA with Ribozero kit (Illumina). The libraries were sequenced on the Illumina Novaseq platform with a read length of 100 nt in paired end configuration. Grant ID: 20-SSP-114 Funding agency: Searle Scholars Program