UC Santa Cruz

ABSTRACTNon-Coding RNAs Regulate Innate Immune Signaling Haley Lynne Halasz As our understanding of the human genome has progressed, so has our interest in a class of molecules that challenge the central dogma of biology: DNA is transcribed into mRNA, mRNA gets translated into protein. These molecules have become known as “Non-coding RNAs” because they carry out cellular functions as RNAs without coding for proteins. One of the many compelling things about non-coding RNAs, is that their expression is highly context and tissue specific. The work presented here, focusses on a specific class of non-coding RNAs, long non-coding RNAs (lncRNAs), and how they function in the context of innate immunity and inflammation. Chapter 1 reviews the importance and clinical implications of lncRNAs in inflammatory diseases. Chapter 2 describes a highthroughput CRISPRi screening approach to identifying lncRNAs that regulate a prominent inflammatory signaling pathway, the NFkB pathway, in human monocytes. Chapter 2 also focusses on uncovering the mechanism of one such lncRNA, LOUP (lncRNA originating from upstream regulatory element of SPI1 [also known as PU.1]). Our current understanding of lncRNAs that regulate inflammatory signaling in human monocytes is quite limited. Monocytes are precursors to macrophages, and both are critical effector cells of the innate immune system. Monocytes and macrophages are some of the first cells to respond to pathogens and are characterized by their ability to phagocytose. As presented in Chapter 2, we have used a human monocytic cell line (THP1 cells) as a model system to conduct a reporter based CRISPRi screen to identify lncRNAs that regulate NFkB signaling. NFkB is a transcription factor that activates transcription of hundreds of inflammatory genes. Dysregulation of this pathway underlies many diseased states. Our screen successfully identified numerous lncRNAs that regulate NFkB positively or negatively. One of the topmost significant candidates was a previously described lncRNA, LOUP that neighbors the myeloid lineage determining factor SPI1. In addition to driving myeloid differentiation, SPI1 is a transcription factor known to also control activation of inflammatory genes. Interestingly, we found that when we knockdown LOUP with CRISPRi, the TLR4/NFkB-driven inflammatory response is broadly upregulated, designating LOUP a negative regulator of NFkB. Previously, it’s been found that the lncRNA LOUP transcript directly mediates interactions between an upstream response element (URE) and SPI1’s promoter, hence regulating transcription of SPI1. Consistent with this previous work, we also found that expression of LOUP enhances SPI1 expression, but that this does not account for LOUP’s inflammatory regulation. Remarkably, knowledge of the complexity of the human genome continues to develop, and it's now appreciated that some designated “non-coding” RNAs produce very short functional peptides. Upon further investigation of LOUP’s coding potential, we discovered that it produces a small peptide responsible for LOUP’s ability to negatively regulate NFkB. The studies described in Chapter 2, reveal new insights for lncRNAs and short ORF-encoded peptides (SEPs) in the context of inflammation. Relatively little is known about the mechanisms underlying how lncRNAs function in this context, let alone SEPs. While some lncRNAs have been identified as regulators of inflammation, the proportion of these genes that have been ascribed functions is still quite small, and the ability of some of these genes to produce short peptides has only been recognized somewhat recently. To our knowledge, this is the first successful CRIPSRi lncRNA screen performed in monocytes, making this not only a great technological advancement, but an invaluable resource. Using reliable high-throughput methods to screen for functional lncRNAs genome-wide is a highly efficient way to identify and further study the mechanisms of this under-examined class of genes.