Advances in next-generation sequencing and ribosomal profiling methods highlight that the proteome is likely orders of magnitude larger than previously thought. This expansion potentially occurs through translational recoding, a process that results in the expression of multiple variations of a protein from a single messenger RNA. Our laboratory demonstrated that cyclooxygenase-3/1b (COX-3/1b), a frameshifted, intron-1-retaining, alternative splice variant from the COX-1 gene, is multiply recoded, which results in the translation of at least seven different COX-3 proteins. Two of the recoded COX-3 proteins that we identified are active prostaglandin synthases and are inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). Here we show that the other non-prostaglandin-generating recoded COX-3 proteins perform new roles in innate immunity, a process in which COX are known to generally function. Our analyses determined that these recoded COX-3 proteins bind at or near the amino-terminal region of ATG9a, a critical regulator of both canonical (i.e. digestive autophagy associated with mTORc inhibition and nutrient deprivation) and non-canonical (i.e. xenophagy involved in the innate immune response to invading organisms) autophagy. We further show that this process requires mTORc signaling activity, which opposes the digestive pathway. As a final confirmation of the biological relevance of these recoded COX-3 proteins and their central role in xenophagy, we demonstrate that expression of these COX-3 proteins in an encephalomyocarditis virus infection model system differentially affects infectious virion production. These COX-3 proteins also associate with recoded cytosolic nucleobindin around large, innate immune-related, large LC3-II positive structures (LLPSs). Through mutagenizing catalytic residues of recoded COX-3 proteins and drug assays, we determine LLPS formation is dependent on oxylipin generation.

To increase diversity of enzymes and proteins, cells mix and match exonic and intronic regions retained in mature mRNAs by alternative splicing. An estimated 94% of all multi-exon genes express one or more alternatively spliced transcripts generating proteins with similar or modified functions. Cyclooxygenase is a signaling enzyme that catalyzes the rate-limiting step in the synthesis of diverse bioactive lipids termed prostaglandins. Prostaglandins are involved in myriad physiological and pathopysiological processes including vasoregulation, stomach mucosal maintenance, parturition, pain, fever, inflammation, neoplasia and angiogenesis and are inhibited by aspirin-like drugs known as NSAIDs. In 2002 an alternatively spliced, intron-1 retaining variant of COX-1 was cloned from canine brain tissue. This new variant, termed COX-3 or COX-1b, is an enzymatically active prostaglandin synthase expressed at relatively high levels in a tissue and cell type dependant manner in all species examined. In humans and most rodent species intron-1 is 94 and 98 nucleotides long respectively. Retention of the intron in these species introduces a frameshift and is predicted to result in translation of a very small 8-16kD protein with little similarity to either 72kD COX-1 or COX-2, calling into question the role of this variant. In this dissertation, I present my results from cloning and ectopically expressing a complete and accurate COX-3 cDNA from both rat and human. I confirmed that COX-3 mRNA encodes multiple large molecular weight cyclooxygenase-like proteins in the same reading frame as COX-1. Translation of these proteins relies on several recoding mechanisms including cap-independent translation initiation, alternative start site selection, and ribosomal frameshifting. Using siRNA and Western blotting I have identified some of these proteins in tissues and cells. Two COX-3 encoded proteins are active prostaglandin synthase enzymes with activities similar to COX-1 and represent novel targets of NSAIDs. Other COX-3 proteins have unknown function, but their size and cellular location suggest potential roles as diverse as cytosolic enzymes and nuclear factors.