Elucidating the role of long non-coding RNAs in the innate immune response: Identification of functional domains that regulate inflammation

Upon completion of the human genome project, the biggest surprise was that humans have far fewer genes than previously expected (~ 19,000) and that this is coded by less than 2% of the available DNA. Significantly, recent studies have suggested that much of the remaining 98% of DNA is turned into 'non-coding RNA' and that these are important regulators of gene expression. For convenience, we commonly divide this non-coding RNA into small non-coding RNAs and long non-coding RNAs. At the present time, we have little idea about the role of the majority of this non-coding RNA although recent studies have indicated that these may regulate the immune response.

In order to fight infection by bacteria, fungi and viruses, the body has a complex defense system called the immune response. This involves the recognition of these microorganisms and release of a range of chemicals that recruit and activate immune cells (inflammation) that are involved in removing these un-wanted invaders. Under normal conditions, this inflammatory response is then switched off. However, under certain circumstances, prolonged activation can be life-threatening or lead to the development of common conditions such as asthma, diabetes, cancer and cardiovascular disease. For this reason, it is important to understand the mechanisms that regulate both the activation and inhibition of this immune response.

Previous studies have shown that the immune response is controlled by a family of small non-coding RNAs called microRNAs. Significantly, we have preliminary evidence showing that long non-coding RNAs might also be novel regulators of innate immunity, specifically by controlling the release of the chemicals used to kill the invading microorganisms. In this project, we will extend these studies by identifying those long non-coding RNAs that regulate this inflammatory response in both human and mouse cells. Using this information, we will then identify and validate the sequences that are important in mediating their actions. Overall, these investigations will help us understand the role of long non-coding RNAs as regulators of inflammation and the immune response to invading pathogens.

The innate immune response is responsible for the recognition of invading pathogens and their subsequent removal through induction of an inflammatory response. To avoid unwanted inflammation, organisms have evolved multiple mechanisms that regulate the innate immune response. Significantly, as part of a previous BBSRC project grant (BB/K006223/1), we have preliminary evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of this inflammation. Thus, we have identified 32 lncRNAs that are induced in multiple cell types following activation of innate immunity, of which 5 (to date) have been shown to regulate inflammation.

Despite these early indications of their importance, little is known about either the numbers of lncRNAs that regulate the innate immune response nor their mechanism of action, although it is speculated that this is mediated through conserved domains. In this project, we will address these important issues by initially using next generation sequencing in combination with knockdown studies, to produce a catalogue of lncRNAs that regulate the inflammatory response following activation of innate immunity in humans (Objective 1) and mice (Objective 2). From these catalogues, we will shortlist 4 conserved lncRNAs that will be subjected to detailed mechanistic analysis (Objective 3). Importantly, as this functional and mechanistic data is collected, bioinformatics and experimental approaches will be applied to identify and confirm the existence of any conserved sequences (domains) that mediate the action of lncRNAs in the inflammatory response (Objective 4). Overall, these studies will provide both by a quantitive (lncRNA catalogues in humans and mice) and qualitative (lncRNA mechanism and conserved domains) leap in our understanding of lncRNA biology, resulting in high impact publications. This information will also be important to the future production of knockout/transgenic mice for examination of lncRNA function in vivo.

Academic Impact

The work outlined in this proposal will examine the role of a novel family of non-coding RNAs in inflammation and the innate immune response. Importantly, we also intend to be the first to identify the domains that regulate the activity of these immune-modulatory lncRNAs, thereby providing fundamental information about the mechanism of action of these novel mediators. From an academic point of view, the results of these studies will benefit scientists working in the area of immunology, RNA biology and those engaged clinical work related to inflammation.

Economic and Societal Impact

Inappropriate activation of the inflammatory response is associated with multiple diseases including asthma, rhinitis, eczema, rheumatoid arthritis, coeliac disease and systemic lupus. For this reason, there is considerable interest amongst biotechnology and pharmaceutical companies in the identification of novel anti-inflammatory drug targets. A number of companies including Santaris (Roche), Regulus, Alynlam and ISIS pharmaceuticals are currently undertaking clinical trials (Phase 1-3) with oligonucleotide based therapeutics (antisense and siRNA) that target mRNAs and miRNAs implicated in a range of diseases including cancer, asthma, viral infections and high cholesterol. Importantly, an identical approach could be used to target the activity and/or expression of lncRNAs and thereby permit development of new drugs for the treatment of inflammation. Indeed, a Boston based start-up company called RaNA therapeutics (http://ranarx.com) has recently raised $55M to develop a locked nucleic based antisense approach to modulating the activity of a lncRNA, which results in the induction of protein expression. Clearly, the development of novel anti-inflammatory drugs will be of great benefit to the patients suffering from these diseases.