RNA, RNA Everywhere

The enhancers that drive transcription are themselves transcribed, in a regulatory process.

The last decade or two have not only brought a genomic revolution in molecular biology, but also remarkable discoveries in RNA, finding micro RNAs, conserved long non-coding RNAs, piRNAs, siRNAs, snoRNAs, and now eRNAs, for enhancer RNA. Even though most of the genome is junk and remains junk, 80% of it is transcribed, so the cell turns out to be a flurry of all sorts of incredibly diverse RNAs beyond the classic molecular biology trinity, which is: mRNA to carry the gene sequence from the DNA, tRNAs that serve as the plug-in adapters between triplets on those mRNA messages and the amino acids they will become in the protein, and the rRNA that forms the body and catalytic core of the ribosome, operating the converyor belt that brings together the first two RNAs to synthesize proteins.

In retrospect, we perhaps should not have been so surprised, since RNA has been there from the most ancient period of life, and the messiness of biology tends to elaborate complexity, using any wrinkle or handle as a regulatory process. But for a couple of decades we were blinded by the preponderant relative mass (and, to be fair, importance) of the RNA trinity in the cell, and only recently have we had the technical means to find the great diversity lurking beneath.

A recent review catalogues the findings and hypotheses about the newest member of this tribe, eRNA, in detail. In eukaryotes, especially as they become more complicated, genes are driven by quite elaborate collections of "enhancers", which are DNA segments typically far upstream, by thousands to hundreds of thousands of base pairs, that bear a cluster of DNA binding sites where regulatory proteins bind, which either turn that gene off or on. One gene may have many separate enhancers, each typically devoted to one phase of development and/or one location in the body where it drives the activity of its target gene.

Schematic of gene control, showing an enhancer (LCR) that has several colored regulatory proteins bound to it. At the same time that it loops through space to contact its target (ßmaj gene), it is also transcribed to short RNAs (red) by RNA polymerase (P). The small discs all over the place are histones (H), which are modified with various colored methyl and acetyl groups in another regulatory process.

Enhancers can do this because they form loops from their distant sites, to contact the start point of their target gene, at what is called the promoter (pictured above as a bold elbow+arrow, when active). This arrangement means that it hardly makes much difference how far away the enhancer is- the proteins it binds can ignore the many kilobases, sometimes hundreds of kilobases, of linear distance in the DNA between themselves and the target gene's start site. But it also means that there needs to be some way to "insulate" one gene and its gaggle of far-off enhancers from those of other genes, which one wouldn't want crossing over into each other's territory and turning each other on. That is a story for another time.

The new and quite paradoxical finding is that enhancers are themselves transcribed, and that these resulting eRNAs are not just accidental junk, but play a significant role in the operation of the enhancer and the regulation of its target gene. As pictured above, (in red), eRNAs come streaming off the enhancer long before the target gene gets turned on. And if those eRNAs are degraded by an experimenter's intervention, typically (and ironically) by programming siRNAs against them, then the target gene turns on much less than otherwise. So it is not just the act of enhancer transcription that is important, though that is thought to have some regulatory effects as well, but the products themselves, at least in some cases.

eRNAs are thought to interact with another level of regulation, which operates through the histones which typically package all eukaryotic DNA. Any protein that binds to a specific site needs to get through this packaging, which can happen in some cases by detecting the DNA on the outside of the histone, or by waiting for a stochastic loosening of the histone from the DNA. But after the pioneer proteins find their sites, they can attract other regulators that specifically modify lysines (K) on the histone with methyl and ethyl groups, neutralizing their charge and lowering their binding affinity to the negatively charged DNA. This process "opens" up the chromatin for other regulatory proteins to bind. The specific lysines that are modified on histones constitute a complex code that marks areas in chromatin for various stages of transcriptional and other activity. The eRNAs have yielded mixed behavior in this pathway, sometimes being required for histone modification at target genes, though not typically at the enhancer region.

Much is still unknown about these eRNAs- how general their occurrence is, how they work, what these little RNAs are doing in the enhancer-promoter complex, and what drives their own transcription. It is like wheels turning within wheels, within wheels- where does the gene activation process ultimately begin?

• Bonus reference on eRNA.
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