PROJECT SUMMARY/ABSTRACT The mammalian immune system is critically dependent on the activity of two sister receptors, RNase L and Ire1, which provide innate immune defense against viral double-stranded RNA, and ensure production of secreted proteins such as antibodies and mucins. Recent work reveals that these two receptors employ a unique strategy not used by any other signaling proteins: cleavage of messenger RNA (mRNA). The goal of the proposed work is to decipher the cellular mechanisms of mRNA regulation by RNase L and Ire1 in human lung epithelial cells. These cells are ideally matched to the biological questions under study due to their high protein secretion and innate immune activity, as well as strong activity of both RNase L and Ire1. The central method that will be used and further developed in the proposed work is a new RNA-seq-based protocol, LRtcB-seq, which allows for RNase L and Ire1 cleavage sites in mRNA to be specifically captured and mapped with single-nucleotide resolution. It was recently demonstrated that RNase L uses global mRNA cleavage to reprogram translation, in a mechanism that favors innate immune protein synthesis. Preliminary data now suggest there is an additional layer to this mechanism; ribosomes become stalled on abundant 2’,3’-cyclic phosphate ends in mRNA produced by RNase L activity. It is further posited that ribosome stalling contributes to the rapid translational arrest enacted by RNase L, which is then corrected by the key ribosome rescue factor Pelota to enable immune defense protein synthesis. Aim 1 of this proposal focuses on interrogating ribosome stalling by cyclic phosphates using LRtcB-seq, and to determine the role of Pelota in resolving ribosome stalls and promoting innate immune defense. Although RNase L and Ire1 function in separate pathways, growing evidence indicates Ire1 too can execute broad mRNA cleavage to modulate cell function. This process, termed regulated Ire1- dependent decay (RIDD), is the least defined activity of Ire1, particularly for human cells. Aim 2 of this proposal addresses the key question in the RIDD field: which mRNAs, cell-wide, are the direct targets of Ire1. The proposed LRtcB-seq-based approach is both sensitive and unbiased, overcoming a critical deficiency in current literature. The comprehensive mapping of mRNA cleavage targets will advance mechanistic understanding of the cellular functions of RNase L and Ire1, and lay necessary foundations for developing therapies for infections and protein folding diseases. The proposed work brings together the lab of Alexei Korennykh (Princeton), who has years of experience in mechanistic and cell biology studies of RNase L, and the lab of Peter Walter (UCSF), who is a key co-founder of the Ire1 field. The collaboration between these labs will provide the foundation for achieving the aims of the proposed work and for scientific development of the trainee.