Perhaps it won’t be long before everyone, critics included, looks at the “junk DNA” concept in the rear-view mirror. In Nature Methods this month, the editors give voice to paradigm shifters. “The research community focused on noncoding RNAs keeps growing,” writes journalist Vivien Marx in a Technology Feature (open access). But “Skepticism about the field has some history.” Like dark horse candidates taking the lead, the treasure hunters appear to be way ahead of the pack. Revealingly, the headline is written in past tense: “How noncoding RNAs began to leave the junkyard.”

Junk. In the view of some, that’s what noncoding RNAs (ncRNAs) are — genes that are transcribed but not translated into proteins. With one of his ncRNA papers, University of Queensland researcher Tim Mercer recalls that two reviewers said, “this is good” and the third said, “this is all junk; noncoding RNAs aren’t functional.” Debates over ncRNAs, in Mercer’s view, have generally moved from ‘it’s all junk’ to ‘which ones are functional?’ and ‘what are they doing?’ Researchers are mapping out the future of the field, which is the theme of a second story in this issue. Scientists in the ncRNA field have faced skepticism and worked to dispel it. [Emphasis added.]

Vivien Marx lists some “exemplar” ncRNAs that have been shown to be functional:

1. Xist: this lncRNA inactivates one of the X chromosomes in female mammals.
2. The lac operon is part of a gene regulatory circuit required for the transport and metabolism of lactose in E. coli and in many other enteric bacteria.
3. micF was among the first regulators of gene expression discovered. It inhibits translation of a target messenger RNA in response to environmental stress.
4. lin-4, a microRNA (miRNA), affects expression of a messenger RNA (mRNA) after it has been transcribed.

As Aurora Esquela-Kerscher from Eastern Virginia Medical School points out, lin-4 is “the founding member of the miRNA superfamily.” Many miRNAs have been identified in plant, animal and viral genomes, and they appear to affect diverse cellular processes including proliferation, apoptosis, differentiation, metabolic and immune responses. Studying lin-4 in C. elegans brought a fundamental understanding of miRNAs mechanisms. miRNAs are “more complex than initially predicted,” and they direct important functions in the nucleus and cytoplasm; they modulate genes in positive and negative ways. “Stay tuned — these tiny RNAs likely have bigger surprises in store for us!”

Noncoding RNAs are ubiquitous in the cell. Some act like switches that turn gene expression on and off in different cell types and tissues and in different stages of development.

Once researchers discovered that one miRNA can regulate hundreds of different mRNAs — this began with the work on lin-4 — “it was a total game changer,” says Linscott. “Suddenly, we had an explanation for how many different parts of a given pathway might be influenced by a single noncoding element.”

History and Outlook

Marx’s article takes readers through a brief history of RNA discoveries, from the year 1869 when nucleic acids were first identified to the present. The field still lacks maturity, however, so design advocates should not rush to assume every base in the genome is functional. When the ENCODE consortium found that 80 percent of the genome was transcribed, it did not imply that the functions of all those transcripts were understood.

What was lacking then and what is lacking still, he says, is a theory that would allow fitting RNA into the larger scheme of regulation. “Because all of the examples we knew of were kind of one-offs,” says Guttman. Small nuclear RNAs, for example, will base-pair with introns at splice sites to guide the splicing machinery. “How do you generalize beyond splicing?” he asks. Small nucleolar RNAs base-pair with 45S pre-ribosomal RNA; that’s another “one-off.” Xist, a lncRNA, silences one of the two X chromosomes in female mammals’ X chromosomes, and it presents another extrapolation challenge, says Guttman. The evidence about Xist is generally accepted, he says, but it remains seemingly exceptional.

Genomics has clearly “evolved” beyond its earlier protein-centric thinking, Marx concludes, but “Doubts may remain and some aspects remain challenging to prove.” Design advocates should avoid the appearance of repeating “just-so stories” about functions of ncRNAs. Maite Huarte advises, “The lack of rigor in some studies has fed the skepticism of some researchers, and we face the challenge of producing the best possible evidence to overcome this prejudice.”

A month earlier in The Scientist, Christie Wilcox wrote about “The noncoding regulators in the brain.” Her article features a helpful infographic about types of ncRNAs. They are “not so noncoding” after all:

Noncoding RNA may be a bit of a misnomer. At least some lncRNAs, circRNAs, and transcripts of other so-called noncoding genomic regions do, in fact, contain open reading frames that code for micropeptides.

The coding-noncoding nomenclature for RNAs arose in the early days of genomic sequencing. “That’s kind of human nature there, to have to compartmentalize everything,” says University of Queensland molecular neuroscientist Timothy Bredy. But when it comes to the diversity of forms RNAs can take, researchers now know that such restrictive boxes just don’t capture reality. “We have to come up with a new way to describe them — like multidimensional, or multifunctional RNA species,” he says.

The Road Ahead

In a companion article in Nature Methods, Marx looks to “Some roads ahead for ncRNAs.” Now that GENCODE has identified 20,000 lncRNAs and the FANTOM consortium has identified 30,000, the priority is to “develop and apply methods to identify and understand the roles of lncRNAs and RNA networks” so that the scientific community can announce clear-cut results and propose applications.

As matters shift from sweeping statements about junk and transcriptional noise, tasks shift to the practicalities of exploring functionality of ncRNAs to uncover their roles in differentiation, development and disease, says Mercer. He sees a new generation of scientists settling in to do the “hard work” of building on the field’s accomplishments, in which technology development and application have mattered. It will matter, for example, to combine methods — existing ones and new ones still to be developed.

While there is “no dearth of ncRNAs” (the human genome has 96,411 lncRNA genes and 173,112 lncRNA transcripts, Marx notes), untangling the intricacies of the genomic network will take time. Alternative splicing can affect ncRNAs, producing different functions, just like it does with protein-coding transcripts. But since knockout experiments with ncRNAs are not as definitive as those with genes, one must not generalize from observations too quickly. What happens in one cell type may operate differently in another. John Mattick of the University of New South Wales is optimistic about the functional capacity of the genome:

Genomes, says Mattick, are “zip files” of transcription, with many layers of information. “The human genome is incredibly information dense,” he says. ncRNAs are, for example, involved in brain development in ways yet to be deciphered. “There’s just a whole world of these things that are being produced in different stages of differentiation and development, and we’ve hardly scratched the surface of which ones do what.”

Gene Yao at UC San Diego views the genome as a collection of isoforms involving both genes and non-coding regions. Seen that way, there is far more information packed into the genome than the small number of genes that surprised biochemists when the Human Genome Project was completed.

Yeo advises keeping in mind that beyond the around 25,000 human genes there are hundreds and thousands of alternative isoforms. “When I think about RNA, I think really isoforms,” he says. Because isoforms cannot be distinguished by in situ hybridization, “I would say we’re missing 80% of the picture,” such as subcellular effects. This adds to the live cell measurements that aren’t readily possible.

Bringing Critics Around

There are still critics who think the RNA research community is “overstating the extent of lncRNA function” and making unsubstantiated claims. The exemplars cannot yet be generalized to other ncRNAs of unknown function, they argue. Proponents respond that progress is hard. It is much more difficult to perturb ncRNAs without affecting anything else. Non-coding RNAs are not “evolutionarily conserved” like many genes are — a clue geneticists have used to evaluate likely functional importance. Each ncRNA must be evaluated within its cell type and developmental context. 

Some critics point to the low expression levels of ncRNAs to argue that they can’t be all that important. Marx lets Mitch Guttman of Caltech respond; he points out that “lncRNAs ‘can punch above their weight,’ and act in a nonstoichiometric way to amplify effects.” By analogy, one switch can turn a lot of things on or off. Wilcox quotes Duke University developmental neurobiologist Debra Silver:

“It’s sort of like hitting, for lack of a better term, almost a master regulator of gene expression,” says Silver. “And by doing that, it’s going to influence gene expression of its targets likely in a very cell-specific, tissue-specific, timing-specific fashion, and that itself could then affect expression of downstream targets below that.” Because of this, she adds “even though our genomes of human and, say, chimpanzees are remarkably similar globally at the DNA level, there is a whole host of regulatory changes at the RNA level that are likely to contribute synergistically to human-specific traits.”

Wilcox agrees that RNAs have gotten “a lot more attention” in the last 10 to 15 years, but the field needs to move past the cataloging stage and determine functions of the thousands of identified ncRNAs. “One of the trickier aspects of studying noncoding RNAs is that they don’t act alone,” she says. “Rather, they function in networks and systems in cells that can be tricky to recapitulate in experimental models.”

These are examples of debates currently going on in the field of RNA research. Now that voluminous data has been collected on noncoding regions of the genome, researchers are busy doing the hard work to analyze it and understand it. More young scientists are choosing RNA research as their career specialty. Without overstating the case, design advocates can watch this scientific revolution with anticipation that the degree of specified complexity in the genome will continue to grow.