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.