Observations on Regulatory RNAs 2014

The last two decades have been exciting times as simplistic genomic models were updated to explain mechanistic details. Along the way, we discarded the expectation that sequencing the genome would lead to quick understanding of the structure‒activity relationship (SARs) of life. Given the proper tools, biochemists and biologists are discovering that the SARs responsible for regulation of living systems are exceedingly complex. Ribonucleic acids (RNAs) are much more varied than the limited model involving messenger RNA (mRNA) and transfer RNA (tRNA) of the 1990s and before.

One example is the proceedings of the Regulatory RNA Conference, which attracted more than 330 scientists to the Claremont Hotel in Berkeley, CA, from October19 to 21, 2014. I was introduced to long and short noncoding RNA, cyclic dinucleotides, RNA-mediated genome rearrangement, post-translational modification of RNA including methylation, chiral RNA, CRISPR, and more.

RNA-guided DNA rearrangement

In the opening session, Prof. Laura Landweber of Princeton University, NJ, described the most amazing genome rearrangement, which is orchestrated by RNA. The ciliate Oxytricha possesses two genomes. One is the germ line genome, which is about 20 times larger and very complex. During development, Oxytricha forms a smaller “nanochromosome” inside the protozoa by eliminating nearly all the noncoding DNA. It arranges the short pieces of coding DNA segments to produce intact genes. The product is a mature genome with about 16,000 nanochromosomes ranging in size from 400 to 66,000 base pairs that code for a single protein or RNA. Long noncoding RNAs guide the entire rearrangement. Further, the long coding RNA provides continuity across several generations, including remodeling and DNA repair. Prof. Landweber noted that the process bypasses the traditional mode of inheritance via DNA.

As with DNA and proteins, methylation is a common modification of the biopolymer that affects activity. Add RNA to the list. Tao Pan of the University of Chicago, IL, pointed out that methylation of RNA is but one of about 100 post-translational modifications that control its function. In RNAs, methylation of adenosine produces N6-methyl adenosine (m6A). Several lectures discussed the role of m6A. Suffice it to say, it appears to be the most common derivative of RNA, but some of its roles are just now coming into view. Pan went on to discuss how RNA binding proteins control the activity of long RNA by binding at particular binding motifs (PBMs). Many of these motifs are buried in the RNA structure. The exact mechanism of interaction and control is not clear, but proximity plots show that activity often falls off as the distance in bases increases. However, activated states may include bending and folding to bring certain PBMs close together. Methylation can facilitate structural changes, which improves access to heteronuclear ribonucleoprotein C (hnRNP C). Reducing methylation reduces the interaction of hnRNP C to the 16,000 of the potential 39,000 potential sites. Pan’s lab has identified over 2700 m6A switches for hnRNP C binding among a wide range of cellular processes.

Methylation of cytosine in DNA is important in controlling transcription. Steve Jacobson of the University of California at Los Angeles reported that DNA methylation in Arabidopsis thaliana is usually performed by DNA methyltransferase (DRM2). In mammals, methylation involves a similar enzyme called Dnmt3.

C. elegans was a frequent poster child. This poor simple worm is an ideal subject. The genome produces about 960 cells. The worm is transparent and hence compatible with high-resolution scanning. Chemicals with severe impact on cell function often show up as visible abnormalities under the microscope.

Cell regulation often involves timing or almost counting. Prof. Christopher Hammell of Cold Spring Harbor Laboratory, NY, showed that a complex involving BLMP-1 and LIN-42 works as a simple clock with positive and negative feedback which functions as a biological clock or gate. Essentially, the complex responds to a need by producing the desired product, but also simultaneously produces an inhibitor of the same complex, producing pulses of activation followed immediately by suppression of gene transcription.

L-RNA

I’d not thought of it before, but of course, RNA is chiral. Indeed, all-natural RNA is made from the D-enantiomers. Prof. Gerald Joyce of The Scripps Research Institute (La Jolla, CA) pointed out that L-RNA has several properties that make it attractive for special situations, particularly for stable, high-affinity aptamers. In contrast to D-RNA, L-RNA is not degraded by nucleases, which can improve pharmacokinetics. Prof. Joyce’s lab has made aptamers that bind to D-RNAs (mir-21, mir-225, and mir-10b with kd of 25, 19, and 47 nm, respectively). Plus, L-RNA seems to be nontoxic and does not bind human DNA with Watson-Crick pairing.

Clustered regularly interspaced short palindromic repeats (CRISPR)

Genome editing with CRISPR-Cas technology is used for sequence-specific genome (DNA) cleaving and editing. The process is controlled by guide RNAs that direct Cas9 endonuclease to specific sites. The exact mechanism of how the Cas9 guide RNA works is not yet understood, but the presence of a bulge in the guide RNA appears to be necessary in vivo and in vitro. Nevertheless, Prof. Rodolphe Barrangou of North Carolina State University, Raleigh, has cataloged RNA guides and distinct nucleases to open new options for rational design in molecular biology.

Cyclic dinucleotides

Cyclic dinucleotides (CDNs) are new, robust, small molecules used by bacteria for signaling. In mammalian cells, they are agonists, generally activating STING, which leads to interferon (IFN) response. Prof. Jennifer Doudna of the University of California at Berkeley lectured on the origin and function of CDNs. In mammals, cGAS synthesizes a unique cyclic dinucleotide of adenosine and guanine but with 2’‒3’ linkage, rather than the more common 3’‒5’ linkage.

What is responsible for this unusual linkage specificity? How is it controlled? Prof. Doudna’s group started with a cGAS enzyme from Vibrio Cholerae DncV. Guided by X-ray crystal structures, they found evidence that the order of cyclization in prokaryotes and mammals was the opposite order. Further, by modifying the active site in mammalian enzyme, they could reprogram the order of synthesis of the human site to produce the 3’‒5’ isomer. The correct isomer was confirmed when the reprogrammed enzyme selectively stimulated STING in mammalian cells. Prof. Doudna predicted that this exercise may lead to a new class of small-molecule drugs. On a side note, I was surprised when Prof. Doudna mentioned that the analytical procedure for the linkage isomers of CDNs was thin layer chromatography (TLC)—old fashioned, but hey, if it works…

The state-of-the-art in regulatory RNAs

The reports above cover a variety of topics, with the only focus being regulatory RNAs. With the exception of CRISPR above, I’d characterize the subject matter as descriptive chemistry. Indeed, it appears that the state-of-the-art is one PI mining a particular niche. This reminded me of the biochemistry lectures I attended 50 years ago, when the MO was each PI focused on characterizing one protein. The focus on descriptive chemistry is an essential early stage in development of a field. Indeed, the vast majority of references cited in the lectures and posters were dated within the last five years. I expect that regulatory RNAs will quickly advance to using the insight gained from mastering the descriptive information to a stage with rapid expansion and beneficial utilization of the technology.

Posters

The lecture program was augmented by almost 200 posters reporting on different aspects of regulatory RNAs. I requested copies of several. I plan to invite some to recast their poster to manuscripts to appear in future issues of American Laboratory.

Exhibition and sponsors

The commercial forum at the meeting was small and focused.

Arraystar (Rockville, MD) promoted kits for arrays optimized for circular RNA. Circular RNA is a novel, noncoding form of RNA produced by a back-splicing process. The circular RNAs are complementary to linear RNA, with high capacity for miRNA binding sites. Hence, they are often called miRNA sponges. Their circular structure reduces miRNA degradation by exonucleases.

Arraystar’s LncRNA is a long-chain (more than 200 bases), noncoding RNA that can bind and block protein-coding genes. The company has compiled a single database from several sources to support research into the structure‒activity relationships. They feel that LncRNAs are candidates for biomarkers of Alzheimer’s and some cancers. Arraystar offered a detailed review of the topic, including cis- and trans-acting LncRNAs.

Biosearch Technologies (Petaluma, CA) focused on Stellaris® RNA FISH probes for locating single or clustered RNAs in cells, tissues, or embryos. The key is to make a fluorescent probe that interacts with RNA in a probe-specific manner. Multiple fluorophores can provide information from co-localized regions. The best results are usually obtained from probes designed to interact with coding regions. Customer guidance is also available for designing probes for variants and introns.

Dharmacon (Lafayette, CO) placed literature in the registration bag. This included a poster of an RNA hairpin with specific events and products organized by year. At the poster’s top was the purchase in 2014 by GE Healthcare from Thermo Fisher Scientific. Fisher Scientific had acquired Dharmacon in 2004. Thermo merged with Fisher Scientific to form Thermo Fisher Scientific in 2006.

Dharmacon offers a broad range of products and services for RNA chemistry, including RNAi for each of the 20,000 genes in the human genome.

Exiqon (Copenhagen, Denmark) specializes in RNA chemistry for research. Products include LNA™ antisense oligonucleotides for inhibition of noncoding RNA. These typically have recognition sites of 12‒16 nucleotides. The LNA is being used to silence two specific genes that are candidate therapeutic targets for neurodegenerative diseases. Exiqon also has a group performing next-generation RNA sequencing on a fee-for-service basis. The list includes sequencing of micro RNA, small RNA, messenger RNA, and whole transcriptome.

Credits

The 2014 meeting of Regulatory RNAs has to be considered a technical success. Accordingly, I’d like to congratulate Jennifer Doudna; Richard Gregory (Harvard Medical School, Cambridge, MA); Cyrus Martin, Editor of Current Biology; and John Pham, Editor of Molecular Cell. The Cell Symposia team deserves thanks for taking care of the human factors that make a networking opportunity really happen. Please monitor the calendar for details on subsequent meetings in this series.

Robert L. Stevenson, Ph.D., is Editor, American Laboratory/Labcompare; e-mail: [email protected].