Functional RNAs?

One of the most important problems in biochemistry & molecular biology is the role (if any) of pervasive transcription. We've known for decades that most of the genome is transcribed at some time or other. In the case of organisms with large genomes, this means that tens of thousand of RNA molecules are produced from regions of the genome that are not (yet?) recognized as functional genes.

Do these RNAs have a function?

Most knowledgeable biochemists are aware of the fact that transcription factors and RNA polymerase can bind at many sites in the genome that have nothing to do with transcription of a normal gene. This simply has to be the case based on our knowledge of DNA binding proteins [see The "duon" delusion and why transcription factors MUST bind non-functionally to exon sequences and How RNA Polymerase Binds to DNA].

If you have a genome containing large amounts of junk DNA then it follows, as night follows day, that there will be a great deal of spurious transcription. The RNAs produced by these accidental events will not have a biological function.

The human genome is large and most knowledgeable biochemists think that 90% of it is junk. There should be a lot of junk RNA produced in any particular cell and if you look at a large number of different tissues you are bound to find that most of the genome is transcribed. In spite of the fact that this is the expected result if you understand the biochemistry, there are those who believe that most of these RNAs have a function—and therefore most of the genome is functional.

The journal Nature Structural & Molecular Biology decided to publish a special issue called Focus on Noncoding RNAs. It came out at the same time as a paper by my colleague Alex Palazzo and his graduate student, Eliza Lee (Palazzo and Lee, 2015). The contrast is remarkable.

Let's look at the Nature Structural & Molecular Biology papers first. Keep in mind that the most important question is whether these RNAs have a function or whether they are just junk RNA produced as a result of spurious transcription. The lead editorial sets the stage [The noncoding explosion].

The long-held view that the primary role of RNA is to code for proteins has been severely undermined. This Focus explores the remarkable functional diversity of RNA in light of recent breakthroughs in noncoding-RNA biology.

In 1958, Francis Crick postulated the 'central dogma' to describe the flow of genetic information from DNA to RNA to protein (Crick F.H., Symp. Soc. Exp. Biol. 12, 138–163, 1958). Experimental evidence then established the mechanistic pathway linking genes to proteins: mRNAs act as transitory templates, tRNAs serve as adaptors between nucleotide and amino acid sequences, and the ribosome functions as the molecular machine that drives protein synthesis. This body of work cemented a canonical view of RNA as primarily a 'coding molecule'. Although tRNAs and rRNAs have obvious noncoding functions, their roles are nevertheless intimately tied to translation, thus reinforcing the notion of RNA as template and structural component to aid in protein synthesis.

The finding that RNA itself is capable of enzymatic catalysis in the 1980s jolted the community and eventually led to the 'RNA world' hypothesis, which proposes that self-replicating RNA molecules were precursors to life based on DNA, RNA and proteins. In comparison, the notion of RNA as a regulatory molecule is relatively recent, and the tremendous number, diversity and biological importance of noncoding RNAs (ncRNAs) are only beginning to be fully appreciated. In this issue, we present a special Focus on noncoding RNAs that explores the functional diversity of ncRNAs, discusses the molecular mechanisms of different RNA interference (RNAi) pathways and highlights the latest breakthroughs in ncRNA biology.

I'm sick and tired of supposedly intelligent people misrepresenting the Central Dogma and misrepresenting the history of a field in order to hype the latest discoveries. You would think that Nature publications would be particularly sensitive to this after the ENCODE disaster.

For the record, regulatory RNAs have been known for 40 years and most of the diverse small RNAs have been around for 20 years. These hardly count as "relatively recent" and it's nothing short of ridiculous to claim that they "are only beginning to be appreciated."

Don't forget that the important question is whether most of these RNAs have a biological function. In other words, SHOULD they be appreciated!

The editors have thought about this question. How do they deal with it?

Thousands of lncRNAs have been discovered to date, but their functional characterization has remained a challenge. This is partly because of a shortage of experimental techniques to explore their functions. In their Perspective, Spitale, Chang and Chu (p 29) highlight recent technological advances that will aid in the functional characterization of lncRNAs and discuss their advantages and caveats. The sheer number and the increasing pace of the discovery of new lncRNAs also present a challenge in terms of lncRNA definition and annotation. This issue is addressed in a Commentary by Rinn and Mattick (p 5), who propose considerations and best practices for identifying and annotating lncRNAs. These guidelines should assist the growing research community embarking on the mechanistic investigation of lncRNAs.

I'm not going to bother discussing either of those papers in any detail. They don't address the question at all. The first paper (Chu et al. 2015) just talks about " ...technologies that have finally made it possible to directly address the where, what and how of lncRNA function..." They assume that most of the RNAs have a function that's just waiting to be nailed down.

The second paper is by John Mattick and John Rinn (Mattick and Rinn, 2015). Asking these guys to write about whether most lncRNAs have a function is like asking Michael Behe to write a critical review of irreducibly complexity. Mattick and Rinn don't discuss the important question at all. They're mostly concerned with how to classify all those thousands of lncRNAs that have been discovered.

If you really want to know about function then you have to read the Palazzo and Lee paper on "Non-coding RNA: what is functional and what is junk?"

They make the same points that have been made repeatedly over the past two decades. Clearly they haven't sunk in and need to be repeated. You begin by assuming, in the absence of evidence to the contrary, that the newly discovered RNAs don't have a function. They are spurious transcripts. That's the default hypothesis. How do you determine if a given RNA has a function?

1. If it's present in significant amounts [see also: How to Evaluate Genome Level Transcription Papers]. We know that the vast majority of RNAs are present at less that one copy per cell. Palazzo and Lee point out that this is a good indication of lack of function although there are situations where low abundance RNA might still have a function.
2. How many have a known function? As of December 2014, there are only 166 lncRNAs with a validated function. (I suspect that not all of them will pan out.) That's after 20 years of looking for function among tens of thousands of putative lncRNAs. It doesn't prove anything but it surely points in one direction.
3. We expect functional RNA to be conserved and most of them aren't. Pallazo and Lee have a good discussion about exceptions to the rule. They are correct to point out that you can have functional transcription without sequence conservations and you can have functional RNAs that have just evolved in one lineage. However, these exceptions cannot account for the thousands of nonconserved RNAs that are supposed to have a biological function.
4. Cell specific transcription. It's often assumed that if an RNA is expressed in only certain tissues, or cells, that this is an indication of function. This is a bad assumption. If we are dealing with spurious transcripts then these will be produced when certain transcription factors bind nonspecifically to DNA. Since different cells have different transcription factors, it follows that they will produce different junk RNAs.
5. What if the RNA is localized within the cell? Palazzo and Lee point out that most of these RNAs are only found in the nucleus and that's where you expect junk RNA. Some are exported to the cytoplasm but that's not a reliable indication of function.

The question has not been answered but if you're a betting person, I'd put my money on most of these RNAs turning out to be junk.

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Chu, C., Spitale, R.C., and Chang, H.W. (2015) Technologies to probe functions and mechanisms of long noncoding RNAs. Nature Structural & Molecular Biology 22:29-35. [doi: 10.1038/nsmb.2921]

Mattick, J.S. and Rinn, J.J. (2015) Discovery and annotation of long noncoding RNAs. Nature Structural & Molecular Biology 22:5-7. [doi: 10.1038/nsmb.2942]

Palazzo, A.F. and Lee, E.S. (2015) Non-coding RNA: what is functional and what is junk? Front. Genet. 6:2. [doi: 10.3389/fgene.2015.00002