A nonprotein-coding DNA region that gives rise to a long, noncoding RNA (lncRNA) has been found to positively regulate transcription of a nearby gene through an enhancer-like cis element. The lncRNA itself appears to be dispensable, at least for this particular function. The finding raises the possibility that other lncRNAs may be incidental. Perhaps even some proteins of uncertain function are incidental to the regulatory activities of their underlying genes. [Joshua Stokes, St. Jude’s Children Research Hospital]
Thousands of our genes produce long noncoding RNAs (lncRNAs), molecules that appear, in many instances, to serve important functions. Yet many lcnRNAs have no known function, leading to speculation that at least some lncRNAs have no biological activity. Yet another speculation, somewhat subtler, is that lncRNAs may be incidental. That is, they may be produced by non-protein-coding DNAs that fulfill biological functions that have nothing to do with the RNAs they express.
Such a non-protein-coding DNA has in fact been identified by scientific team led by Mitchell J. Weiss. M.D., of St. Jude Children’s Research Hospital in Memphis and by Vikram R. Paralkar, M.D., of the Perelman School of Medicine at the University of Pennsylvania. This team determined that one prominent lncRNA appears to be a “red herring,” a molecule that has no evident biological role—whereas the DNA from which it originates does perform an important function, as an enhancer that stimulates the expression of an important protein-coding gene nearby.
The DNA region in question is called Locd. The Lockd RNA product is particularly abundant in mouse red blood cells and some other cell types. To investigate the functions of Lockd, the scientific team led by Drs. Weiss and Paralkar used an advanced gene-editing technique to delete the Lockd DNA from a mouse blood cell line. “When we did this, the expression of Cdkn1b [cyclin-dependent kinase inhibitor 1B] was reduced by 70%,” said Dr. Paralkar.
Next, the researchers used a different technique to block the transcription of Lockd RNA while leaving the Lockd DNA intact. The Cdkn1b expression was not affected. “In other words, getting rid of the RNA transcript doesn't make a difference,” noted Dr. Paralkar, “but getting rid of the DNA does make a difference.”
Details of this work appeared April 7 in the journal Molecular Cell, in an article entitled, “Unlinking an lncRNA from Its Associated cis Element.” The article described how the scientific team studied Lockd (lncRNA downstream of Cdkn1b), a 434-nucleotide polyadenylated lncRNA originating 4 kb 3′ to the Cdkn1b gene.
“Deletion of the 25-kb Lockd locus reduced Cdkn1b transcription by approximately 70% in an erythroid cell line,” the authors noted. “In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by >90% with no effect on Cdkn1b transcription.”
The authors also pointed out that the Lockd promoter contains a DNase-hypersensitive site, binds numerous transcription factors, and physically associates with the Cdkn1b promoter in chromosomal conformation capture studies. In other words, Lockd's transcription-starting “promoter” region contains binding sites for multiple transcription factors. This region happens to lie immediately downstream, on the mouse genome, to Cdkn1b, a gene whose protein product plays a key role in regulating cell division.
The researchers found strong evidence that in the twisted, looping, double-helix structure of the genome, the promoter end of Lockd DNA comes into direct physical contact with the promoter end of its neighbor Cdkn1b, and in that way acts as an enhancer to stimulate Cdkn1b's transcription.
Dr. Paralkar acknowledged that the Lockd RNA may one day be found to have some other function. “It's impossible to prove absolutely that it has no function—but it seems at least that it has no obvious function in regulating its neighbor Cdkn1b,” he said. He emphasized, however, that in determining the function of noncoding DNA and RNA, both DNA-deletion and RNA-blocking experiments—as in this study—are needed to distinguish the function of DNA from its RNA product.
“One has to decouple the transcript from the DNA,” Dr. Paralkar asserted. “Future studies of lncRNA function should adhere to that requirement.”
He added that the discovery of this enhancer function for one example of a lncRNA gene points to the possibility that this is a broadly used mechanism in the genome, found in noncoding and perhaps even some protein-coding genes. Indeed, enhancers are theorized to be one of the key genomic features that distinguish species such as mice and humans–which share nearly all their protein-coding genes, but relatively few of their enhancers and lncRNA-coding genes.
“The fact that mice and humans are so different may be due largely to the fact that their genes are being regulated so differently by enhancers, some of which produce RNA molecules that we detect as lncRNAs,” Dr. Paralkar said.