We previously described the identification of a class of ~3500 conserved large intergenic noncoding RNAs (lincRNAs) using a chromatin signature of active transcription. These lincRNAs are globally functional in the cell and play critical roles in diverse biological processes including in the regulation of the pluripotent cell state. While it is now clear that lincRNAs are functionally important, the mechanism by which they carry out their regulatory role is currently unknown. As many of the lincRNAs interact simultaneously with multiple different protein complexes, one hypothesis is that lincRNAs act as 'flexible modular scaffolds'to bring together protein complexes into larger functional units. In this model, RNA contains discrete domains that interact with specific protein complexes. These RNAs, through a combination of domains, bring individual regulatory components into proximity resulting in the formation of a unique regulatory RNA. Here we propose to decipher the mechanism of lincRNA mediated regulation by understanding how lincRNA-Protein complexes form, localize to regulatory targets, and give rise to phenotypic states. We will address these questions by using genomic methods in conjunction with biochemical methods to characterize lincRNA-Protein interactions, molecular biology to map lincRNAs to their direct targets, genetic methods to delete lincRNA domains and assemble modified RNA genes in vivo, and computational biology to integrate these components into a model of lincRNA mediated genome regulation. We will identify all protein-complexes with which lincRNAs interact, determining where these protein interactions occur on RNA, and determining how these lincRNA-Protein interactions assemble (Aim 1). We will identify the direct regulatory targets of lincRNAs and determine how lincRNAs achieve regulatory specificity (Aim 2). We will determine how individual lincRNA-Protein interaction domains function, and how lincRNAs are functionally assembled by piecing together functional domains (Aim 3). Together, these results will allow us to understand the full complexity of how lincRNAs can utilize discrete domains to target and regulate specific sets of genes and may allow the creation of synthetically engineered RNAs that can carry out engineered regulatory roles.
The recent identification of thousands of large intergenic non-coding RNAs (lincRNAs) represents a missing component in our understanding of genome regulation. Important clues are already pointing to a role for lincRNAs in various human diseases including cancer. Understanding the mechanism by which lincRNAs act will be critical for understanding how they can affect disease states and will provide key insights into therapeutic targeting.
|Munschauer, Mathias; Nguyen, Celina T; Sirokman, Klara et al. (2018) The NORAD lncRNA assembles a topoisomerase complex critical for genome stability. Nature 561:132-136|
|McHugh, Colleen A; Guttman, Mitchell (2018) RAP-MS: A Method to Identify Proteins that Interact Directly with a Specific RNA Molecule in Cells. Methods Mol Biol 1649:473-488|
|Van Nostrand, Eric L; Pratt, Gabriel A; Shishkin, Alexander A et al. (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13:508-14|
|Patil, Deepak P; Chen, Chun-Kan; Pickering, Brian F et al. (2016) m(6)A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537:369-373|
|McHugh, Colleen A; Chen, Chun-Kan; Chow, Amy et al. (2015) The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521:232-6|
|Shishkin, Alexander A; Giannoukos, Georgia; Kucukural, Alper et al. (2015) Simultaneous generation of many RNA-seq libraries in a single reaction. Nat Methods 12:323-5|
|Quinodoz, Sofia; Guttman, Mitchell (2014) Long noncoding RNAs: an emerging link between gene regulation and nuclear organization. Trends Cell Biol 24:651-63|
|Engreitz, Jesse M; Sirokman, Klara; McDonel, Patrick et al. (2014) RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites. Cell 159:188-199|
|Guttman, Mitchell; Russell, Pamela; Ingolia, Nicholas T et al. (2013) Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins. Cell 154:240-51|
|Engreitz, Jesse M; Pandya-Jones, Amy; McDonel, Patrick et al. (2013) The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341:1237973|