STANFORD, Calif. -- The surface of your skin, called the epidermis, is a complex mixture of many different cell types -- each with a very specific job. The production, or differentiation, of such a sophisticated tissue requires an immense amount of coordination at the cellular level, and glitches in the process can have disastrous consequences. Now, researchers at the Stanford University School of Medicine have identified a master regulator of this differentiation process.
"Disorders of epidermal differentiation, from skin cancer to eczema, will affect roughly one-half of Americans at some point in their lifetimes," said Paul Khavari, MD, PhD. "Understanding how this differentiation occurs has enormous implications, not just for the treatment of disease, but also for studies of tissue regeneration and even stem cell science." Khavari is the Carl J. Herzog Professor and chair of the Department of Dermatology.
Khavari and his colleagues have found that, like a traffic cop motioning cars to specific parking spaces in a large, busy lot, a newly identified molecule called TINCR is required to direct precursor cells down pathways toward particular developmental fates. It does so by binding to and stabilizing differentiation-specific genetic messages called messenger RNAs. Blocking TINCR activity, the researchers found, stopped the differentiation of all epidermal cells.
"This is an entirely unique mechanism, which sheds light on a previously invisible portion of the regulation of this process," said Khavari, who is also a member of the Stanford Cancer Institute and chief of the dermatology service at the Veterans Affairs Palo Alto Health Care System. He is the senior author of the research, which will be published online Dec. 2 in Nature. Former Stanford postdoctoral scholar Markus Kretz, PhD, is the first author. Kretz is now an assistant professor of biology at the University of Regensburg in Germany.
Surprisingly, this coordinator extraordinaire is not a protein. (Proteins have traditionally been thought to be the primary movers and shakers in a cell, although that view is now changing somewhat.) Instead, it belongs to a relatively new, and increasingly influential, class of regulatory molecules called long, non-coding RNAs, or lncRNAs. These molecules are so named because they do not carry instructions to make proteins. They are also longer than other regulatory RNAs known as microRNAs.
But even among lncRNAs, TINCR, and its role in epidermal differentiation, is unique.
"This work revealed a new role for regulatory RNAs in gene activation -- by stabilizing select messenger RNA transcripts," said co-author Howard Chang, MD, PhD, professor of dermatology. "This finding highlights the ability of regulatory RNAs to fine-tune gene expression."
The researchers identified the molecule by looking for RNAs that are more highly expressed in differentiating epidermal cells called keratinocytes than in progenitor cells. They found that levels of TINCR (short for "terminal differentiation-induced non-coding RNA") expression were 150 times greater in the keratinocytes. But to figure out what TINCR was doing, they had to develop two new assays: one to help researchers identify interactions between RNA molecules, and another to suss out interactions between a regulatory RNA and its protein partners. Such techniques will become increasingly important as researchers continue to identify the critical regulatory roles played by RNA molecules.
"These long, non-coding RNAs don't have recognizable, classic motifs like proteins do," said Khavari. "And yet, we really need to know with what other molecules they may be physically interacting to truly understand their biological roles."
The first approach, which the researchers termed RIA-Seq, couples an RNA interaction assay with a deep-sequencing technique to identify RNA partners of TINCR. Using RIA-Seq, the researchers found that TINCR and its RNA partners -- many of which encode instructions for proteins essential to the differentiation process -- share a common, short sequence that mediates their binding.
"These conserved, complementary motifs may help TINCR pair up with and stabilize its partner messenger RNAs," said Khavari. "In this way, TINCR may serve as a scaffold for many mRNAs involved in epidermal differentiation."
The second approach used a grid, or microarray, of 9,400 human proteins to which the researchers exposed TINCR. One of the proteins, termed STAU1, bound strongly to TINCR. STAU1 had not previously been implicated in epidermal differentiation, but the researchers found that blocking its activity prevented differentiation in a manner similar to blocking TINCR.
"This effect is quite specific for epidermal tissue," said Khavari, "and it suggests that nature has evolved a simple mechanism to control the tissue-specific expression of a large number of genes. We'd like to understand more about this TINCR-STAU1 complex to get a better idea of how it acts at a biochemical level."
In addition to identifying a unique role for a new lncRNA in epidermal differentiation, Khavari and Chang said they are excited to have developed new tools to understand how these regulatory RNAs function in the cells. "This really helps substantially expand our tool kit that we can use to analyze how RNAs and proteins interact," said Khavari.
Other Stanford researchers involved in the study include senior scientists Zurab Siprashvili, PhD, and Kun Qu, PhD; graduate students Ci Chu, Dan Webster, Ashley Zehnder, Ryan Flynn, Abigail Groff, Grace Kim and Jennifer Chow; and postdoctoral scholars Carolyn Lee, MD, PhD, Ross Flockhart, PhD, Robert Spitale, PhD, and Grace Zheng, PhD.
The research was supported by the National Institutes of Health, the California Institute for Regenerative Medicine and the U.S. Veterans Affairs Office of Research and Development.
Information about Stanford's Department of Dermatology, which also supported the work, is available at http://dermatology.
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