Final Report Summary - FLAME (Long Intervening Noncoding RNAs (lincRNAs): Developmental Functions and Molecular Mechanisms of Action)
RNA is increasingly recognized as an effective therapeutic target. RNA molecules are copied from DNA and used by the cell to make proteins, which are building blocks of the cell. However, thousands of cellular RNAs are not only intermediates for protein production but function on their own as key regulators of diverse biological phenomena. Because an increasing number of RNAs is linked to human pathologies including incurable neurodegenerative diseases, antibiotic resistance, and human diseases caused by RNA viruses such as HIV, Ebola, SARS, influenza, hepatitis C and Zika, RNA molecules represent an untapped source of therapeutic targets. Among various types of RNA molecules, long noncoding RNAs (lncRNAs) have emerged as key regulators of important biological processes including human neurological disorders and cancers. Our research program driven by our interest in RNA biology aimed at understanding the in vivo functions of lncRNAs at the organismal level and dissecting their molecular mechanisms of action. To identify the in vivo functions of lncRNAs, we focused on lncRNAs that are conserved throughout evolution from fish to mammals. We reasoned that lncRNAs under considerable evolutionarily pressure represent a promising set of biologically relevant RNA molecules. All of our studies were conducted in zebrafish and mice as they are amenable to genetic manipulation that allows one, in conjunction with other techniques, to precisely understand lncRNA contribution to normal physiology.
We have found that one of the conserved lncRNAs controls normal animal. Remarkably, this lncRNA controls behavior in adult zebrafish and mice by instructing destruction of another type of regulatory noncoding RNA in the cell called microRNA. We found that degradation of a specific microRNA in particular neurons of the adult brain is required for normal functioning of the cerebellum. If too much of the microRNA molecule is left in the brain, it leads to behavioral alterations in animals, including the impaired motor learning function. In addition, we identified the exact sequence in the lncRNA gene that triggers microRNA degradation. In summary, by demonstrating for the first time the physiological significance of microRNA degradation, directed by a genome-encoded lncRNA, our work resolves a longstanding question in the noncoding RNA field. In addition, our results point to these types of RNA sequences embedded in genome-encoded transcripts as potentially having broad in vivo relevance and serving as therapeutic targets in the future.
Another large achievement of the award was the development of a novel high-throughput technology that enables the identification of RNA–protein interactions in living cells. Because all cellular RNA transcripts are coated by RNA-binding proteins (RBPs) and, in particular, regulatory noncoding RNAs carry out their cellular functions through the diverse protein assemblies in which they are integrated, identifying the protein interaction partners of RNAs is critical to decipher their molecular functions. However, the RBP repertoire reported to bind various RNA transcripts is often skewed toward abundant and promiscuously binding proteins, which can confound functional interpretations. Due to its specific design, our new technology resolved two main obstacles when aiming to identify RNA-bound proteomes: it enabled the identification of proteins associated with RNAs expressed at low endogenous levels, and determined RNA region-specific proteomes allowing the assignment of protein binding to defined regions of a long full-length transcript. Moreover, our method not only identified previously known RNA-protein interactions but also identified novel functionally important RNA-protein interactions that evaded detection with previous methods, reinforcing the advantages of our technology. In summary, we have developed new tools and have established genetic in vivo systems to dissect the functions and mechanisms of action of lncRNAs. Our studies have demonstrated that sequence conservation can guide our understanding of lncRNA functions.
We have found that one of the conserved lncRNAs controls normal animal. Remarkably, this lncRNA controls behavior in adult zebrafish and mice by instructing destruction of another type of regulatory noncoding RNA in the cell called microRNA. We found that degradation of a specific microRNA in particular neurons of the adult brain is required for normal functioning of the cerebellum. If too much of the microRNA molecule is left in the brain, it leads to behavioral alterations in animals, including the impaired motor learning function. In addition, we identified the exact sequence in the lncRNA gene that triggers microRNA degradation. In summary, by demonstrating for the first time the physiological significance of microRNA degradation, directed by a genome-encoded lncRNA, our work resolves a longstanding question in the noncoding RNA field. In addition, our results point to these types of RNA sequences embedded in genome-encoded transcripts as potentially having broad in vivo relevance and serving as therapeutic targets in the future.
Another large achievement of the award was the development of a novel high-throughput technology that enables the identification of RNA–protein interactions in living cells. Because all cellular RNA transcripts are coated by RNA-binding proteins (RBPs) and, in particular, regulatory noncoding RNAs carry out their cellular functions through the diverse protein assemblies in which they are integrated, identifying the protein interaction partners of RNAs is critical to decipher their molecular functions. However, the RBP repertoire reported to bind various RNA transcripts is often skewed toward abundant and promiscuously binding proteins, which can confound functional interpretations. Due to its specific design, our new technology resolved two main obstacles when aiming to identify RNA-bound proteomes: it enabled the identification of proteins associated with RNAs expressed at low endogenous levels, and determined RNA region-specific proteomes allowing the assignment of protein binding to defined regions of a long full-length transcript. Moreover, our method not only identified previously known RNA-protein interactions but also identified novel functionally important RNA-protein interactions that evaded detection with previous methods, reinforcing the advantages of our technology. In summary, we have developed new tools and have established genetic in vivo systems to dissect the functions and mechanisms of action of lncRNAs. Our studies have demonstrated that sequence conservation can guide our understanding of lncRNA functions.