We study the functions of non-coding RNAs in mammalian cells, particularly long ncRNAs (lncRNAs) associated with human cancers. We work with human cancer cells to understand the molecular mechanisms that underlie cell growth, development, and metastasis. Non-coding RNAs have recently been discovered to control many aspects of cellular growth and their interactions with proteins represent a new target area for therapeutics. However, many details of the molecular interactions underlying non-coding RNA-protein interactions are still unclear. The long-term goals of our work are to explain the molecular mechanisms by which RNAs bind and control proteins to perform their functions, to understand how interactions between RNAs and proteins enable assembly of higher order structures and how these complexes act to regulate diverse cellular processes. We aim to control or modulate these interactions to create new functions and prevent or treat disease.
1) Discovery of new lncRNA-protein interactions A number of lncRNAs have been shown to regulate gene expression in eukaryotic cells. We use a stringent method to accurately identify the direct and specific protein interacting factors of an individual RNA molecule, termed RNA antisense purification (RAP). We are using RAP with mass spectrometry to identify the binding partners of functional RNAs in human cells. This information is valuable because we still do not know precisely how lncRNAs bind and coordinate proteins to achieve their functions. Many of the cellular functions of RNAs are predicted to be dependent on protein binding. If we understand their specific interactions, we may be able to control these regulatory events. 2) High-resolution structural characterization of lncRNPs Critical eukaryotic ribonucleoprotein complexes including the spliceosome and the ribosome have been studied in great detail for many years, but little is known about the formation and structure of long non-coding RNA-protein complexes (lncRNPs). These lncRNPs can control epigenetic regulation in female development, gene expression profiles, hormone receptor responses, and many other functions in the cell. We are just beginning to appreciate the roles of long non-coding RNAs in localizing and organizing proteins in the cell in time and space.
3) Revealing new functions of cancer-associated lncRNAs Therapeutics targeting cancer cell growth and development have mainly focused on protein transcription factors and protein-mediated signaling pathways. Recent evidence shows that non-coding RNAs can have a major impact on cancer development and progression. Bioinformatics studies have identified a number of cancer-associated non-coding RNAs whose expression is linked to metastasis and decreased long-term patient survival. A subset of these non-coding RNAs are predicted to act as cancer drivers but their role in regulating cancer cell growth is not well understood. We are currently using a combination of biochemical, biophysical, and genomics tools to examine the functions of these cancer-associated lncRNAs.
Our laboratory team conducts the highest-quality reproducible research and we strive to make science approachable, equitable, and inclusive for all who choose to participate. We value transparency, data sharing, and open access to scientific findings.
Protocol for UV-crosslinked RNA-protein identification: For the most up-to-date RNA-protein identification protocol (RAP-MS), please click the link below. We can provide help and support with optimization for others wishing to use this approach to identify direct RNA-protein interactions.
We are grateful to the University of California and our research sponsors for funding and supporting our work and our people.
McHugh C.A. and Guttman M. 2018. RAP-MS: A method to identify proteins that interact directly with a specific RNA molecule in cells. In Methods in Molecular Biology: RNA Detection – Methods and Protocols.
McHugh C.A., Chen C.K., Chow A., Surka C.F., Tran C., McDonel P., Pandya-Jones A., Blanco M., Burghard C., Moradian A., Sweredoski M.J., Shishkin A.A., Su J., Lander E.S., Hess S., Plath K., and Guttman M. 2015. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521: 232-236.
McHugh C.A., Fontana J., Nemecek D., Cheng N., Aksyuk A.A., Heymann J.B., Winkler D.C., Lam A.S., Wall J.S., Steven A.C., and Hoiczyk E. 2014. A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. EMBO Journal 33: 1896-1911.
McHugh C.A., Russell P., and Guttman M. 2014. Methods for comprehensive experimental identification of RNA-protein interactions. Genome Biology 15: 203-212.
Koch M.K., McHugh C.A., and Hoiczyk E. 2011. BacM, an N-terminally processed bactofilin of Myxococcus xanthus, is crucial for proper cell shape. Molecular Microbiology 80: 1031-1051.
Hoiczyk E., Ring M.W., McHugh C.A., Schwär G., Bode E., Krug D., Altmeyer M.O., Lu J.Z., and Bode H.B. 2009. Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus. Molecular Microbiology 74: 497-517.
Dinglasan R.R., Devenport M., Florens L., Johnson J.R., McHugh C.A., Donnelly-Doman M., Carucci D.J., Yates J.R., and Jacobs-Lorena M. 2009. The Anopheles gambiae adult midgut peritrophic matrix proteome. Insect Biochemistry and Molecular Biology 39: 125-134.
Carra J.H., McHugh C.A., Mulligan S., Machiesky L.M., Soares A.S., and Millard C.B. 2007. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site. BMC Structural Biology 7: 72.
Sergueev K., McHugh C.A., and Hoiczyk E. 2006. Type III secretion systems: Bacterial injection devices for microbe-host interactions. In J. M. Shipley (ed), Microbiology Monographs. Vol. 2: Complex intracellular structures in prokaryotes. Springer, Heidelberg; pp. 339-347.
McHugh C.A., Tammariello R.F., Millard C.B., and Carra J.H. 2004. Improved stability of a protein vaccine through elimination of a partially unfolded state. Protein Science 13: 2736-43.