A new technique developed by researchers at the Stanford UniversitySchool of Medicine allows researchers to identify the exact DNAsequences and locations bound by regulatory RNAs. This informationis necessary to understand how the recently identified RNAmolecules control the expression of neighboring and distant genes. The study offers a startling glimpse into the intricate world ofgene expression and how RNA, once thought to be only a lowlycellular messenger, actively unlocks our DNA-based genome. "We usedto have to just infer where these RNAs were acting based on theirbiological effects," said Howard Chang, MD, PhD, professor ofdermatology.
"But now we can identify precisely where on thechromatin they are binding. We've found that these sites are focal,numerous and sequence-specific." Deciphering the role of regulatory RNAs is critically important tounderstand many cellular functions, including those involved indevelopment, cancer and regeneration. Chang, who is also a member of the Stanford Cancer Institute, isthe senior author of the work, which wias published in Molecular Cell . He and his lab previously identified some of the first knownregulatory RNAs, including one in 2007 they called HOTAIR.
Graduatestudent Ci Chu is the first author of the research. Fifty years ago, researchers first identified messenger RNAs, whichserve to transcribe the genetic instructions encoded in the DNAfrom the nucleus and deliver it to the cell's protein-buildingmachinery. Over time, the unidirectional flow of information fromDNA to RNA to protein became known as biology's "central dogma."Regulatory RNAs challenge that notion by binding to DNA andaffecting which genes are selected to become proteins. The existence of these regulatory RNAs, also called long intergenicnon-coding RNAs, or lincRNAs, was first postulated decades ago byresearchers who realized that DNA packaged into chromatin (a coiledcomplex of DNA and proteins) contains more than twice as much RNAby weight as DNA. They wondered if the RNA coating the chromatinactively regulates when and how specific genes are turned on andoff.
But technical difficulties have made it difficult to identifyprecise sites of binding until now. "To capture this picture, you have to trap that interaction betweenlincRNAs and chromatin in living cells," said Chang. "It was not atall obvious how to do that." In the end, Chu devised an innovative riff on a common assay usedto identify the DNA sequences bound by a class of proteins calledtranscription factors. For that experiment, researchers mix proteinand chromatin and then use antibodies to isolate, orimmunoprecipitate, both the protein of interest and its preferredbinding site.
Instead of using an antibody, Chu and Chang used two or threechemically tagged, complementary nucleotide sequences, or probes,to isolate the regulatory RNAs after they had bound to the DNA.However, they could capture only about 10 percent of the regulatoryRNA using this method. Eventually Chu hit on the idea of usingdozens of individually labeled nucleotide sequences that bindthroughout the molecule. "The problem was that RNA is a long, floppy molecule," said Chang."During the experiment, it would get broken into pieces and thechemical tags would be lost. So, since we didn't know exactly whichportion of the lincRNA binds to the chromatin, we made a series oftagged probes to bind to every part of the lincRNA." This "tiling"approach ensures that, even if the RNA molecule is fragmentedduring the procedure, the researchers will still be able to isolatethe small portion that remains bound to chromatin.
Using the new technique, Chu and Chang were able to investigate thebinding specificity of three lincRNAs - one from the fruit fly, andtwo from mammalian systems. They found that one, called roX2, seemsto coat genes on the single X chromosome in males and facilitategene expression. "We showed that roX2 very precisely binds to the X chromosome onlyin male cells," said Chang, "and it does this in a gradient,binding more and more at the transcriptional end of the gene. Thisappears to help elongation and make transcription more efficient,which is important because male animals have only one copy of the Xchromosome as compared to a female's two." Another lincRNA from humans, a component of the telomerase complexcalled TERC, occupies not just a chromosome's telomeres (protectivecaps at the end of chromosomes), but also genes involved in the Wntpathway, which has been shown to control self-renewal.
"This shows an interesting and direct connection between themachinery for chromosomal replication and self-renewal," saidChang. "It's possible this helps the cell monitor the status of itstelomeres." Finally, HOTAIR, first identified in Chang's lab, seems to recruita gene-silencing protein complex called polycomb to bind broadswaths of DNA. "Taken together, our research shows that our technique is widelyapplicable and can vastly enrich our understanding of howregulatory RNAs unlock the genome in many very specific ways," saidChang. "It's very exciting." Additional References Citations. I am an expert from Luggage, Bags & Cases, usually analyzes all kind of industries situation, such as fleurville diaper bag , pewter clutch bag.
Related Articles -
fleurville diaper bag, pewter clutch bag,
|
