Viroids

Perhaps you've thought that viruses are the simplest sort of "living" thing – if a virus can even be called "alive".

Well, maybe not. Viroids are even simpler. You're on your own as to whether you want to describe them as "alive".

Viroids are not a new discovery – they've been known since 1971. Viroids are found only in plant cells and don't seem to infect animals. They can cause plant pathology, apparently enough to be a serious economic problem.

A viroid consists entirely of a circular piece of RNA, that may be only a few hundred base units long. The smallest known viroid has only 220 units. None of this RNA codes for proteins – unlike virus RNA or DNA, which codes for proteins that (among other things) encapsulate the genetic material. The RNA or DNA of a virus is much larger than the RNA of a viroid. The smallest known virus capable of causing an infection by itself is about 2000 base units.

Viruses reproduce by co-opting machinery of the host cell. The DNA of a DNA virus, for example, is typically normal double-stranded DNA. In the virus life cycle, two separate processes are required (among others). The DNA itself has to be copied with a DNA polymerase enzyme, just as is used in making DNA copies during cell division. The proteins that the virus requires for its coat are also made in the normal way – using RNA polymerase enzyme to make messenger RNA, which can then be used to make proteins in a cell's ribosomes.

RNA viruses are trickier. Sometimes they work by using an enzyme called reverse transcriptase, which makes DNA from RNA. The HIV-1 virus responsible for AIDS is an example. Other RNA viruses, such as human polio viruses, use another enzyme, RNA replicase, which makes copies of RNA directly. RNA viruses usually encode the enzymes that they need for reproduction, to ensure a sufficient quantity of the enzyme.

So how does a viroid reproduce, given that it consists of RNA, but doesn't code for any special enzymes, or any proteins at all? The process isn't well understood, as the following explains:

Viroids: Molecular Vestiges Of The RNA World (5/17/09)

As opposed to plant viruses, which encode proteins that mediate their own replication and movement, viroids depend exclusively on host factors for these purposes. Viroids replicate through an RNA-based rolling circle mechanism with three steps: i) synthesis of longer-than-unit strands catalyzed by a host nuclear or chloroplastic RNA polymerase that reiteratively transcribes the initial circular template, ii) processing to unit-length, which remarkably is mediated by hammerhead ribozymes in the family Avsunviroidae, and iii) and circularization resulting from the action of an RNA ligase or from self-ligation.

Among the many pending issues, how viroids redirect the template specificity of certain host DNA-dependent RNA polymerases to transcribe RNA, is one of the most challenging. In addition, viroids must recruit host factors for their intracelular, cell-to-cell and long-distance movement within the plant. There are also pending questions in this context, the most appealing of which is how members of the family Avsunviridae gain access into the chloroplast; because essentially no other RNA has been reported to traffic inside this organelle, the answer to this question may reveal novel transport pathways in plant cells.

In order to understand what this is saying, the first fact needed is that viroid RNA comes in the form of a circle – no loose ends. In this respect, it is somewhat like bacterial DNA, which consists partly of circular loops of double-stranded DNA, called plasmids.

The interesting part is that viroids are apparently replicated by RNA polymerase, which normally produces RNA from a DNA template, rather than an RNA template. The process is called rolling circle replication, because the enzyme may travel around the loop a number of times, since there are no clear start and stop points. Later, in a separate operation, an RNA enzyme (ribozyme) of the host, cuts the multiply copied segments of viroid RNA back into unit segments, which join at the ends to form a circle again.

What's especially interesting about viroids is that they may give some insight into mechanisms that would be important in the RNA world hypothesis. This is the idea that before proteins existed, or even DNA itself, there was RNA – see here for some recent findings about how RNA itself may have originated.

RNA is capable of carrying genetic information just as DNA does – after all, that's what happens in RNA viruses. The main problem is how it was possible for RNA to reproduce itself. Viroids hardly give us a complete answer to this problem, since proteins (such as RNA polymerase) are still required for replication. But at least, in viroids, we have an example of a replicating entity that consists entirely of genetic information, with no proteins of its own.

Further reading:

Viroids and Virusoids

RNA may form spontaneously

I'd pay attention to this one. Could be a very big story.

Chemist Shows How RNA Can Be the Starting Point for Life

An English chemist has found the hidden gateway to the RNA world, the chemical milieu from which the first forms of life are thought to have emerged on earth some 3.8 billion years ago.

He has solved a problem that for 20 years has thwarted researchers trying to understand the origin of life — how the building blocks of RNA, called nucleotides, could have spontaneously assembled themselves in the conditions of the primitive earth. The discovery, if correct, should set researchers on the right track to solving many other mysteries about the origin of life. It will also mean that for the first time a plausible explanation exists for how an information-carrying biological molecule could have emerged through natural processes from chemicals on the primitive earth.

Here are some more references:

• How RNA got started
• Life’s First Spark Re-Created in the Laboratory
• Origin of life: building an RNA world from simple chemicals
• RNA world easier to make
• Chemists see first building blocks to life on Earth
• New clue to origins of life on Earth
• Molecule of life emerges from laboratory slime

Non-coding RNA and gene expression

Human DNA consists of about 3.4 billion base pairs. A portion of that is actually genes that code for proteins required by human cells – roughly 20,500 genes. (See here.)

However, it's been recognized for a long time that only about 1.5% of human DNA (in terms of base pairs) actually codes for proteins. Little is known about the purpose (if any) of the remaining 98.5%, even though, by some estimates, 80% of human DNA is transcribed into RNA at some time.

This remainder is often called "junk DNA". But it's also known that a lot of it can't really be "junk", and must serve some useful purpose, because the sequences of large portions of it are highly conserved in evolution, being found almost unchanged in the genomes of human ancestors going back hundreds of millions of years.

Some of the 98.5% really does seem to be without useful function, consisting of stuff like transposons, which are DNA sequences that seem to be copied repeatedly and randomly into various parts of the genome (over evolutionary time spans)

The function of other portions of that 98.5% includes such things as introns found within genes, gene regulatory sequences, and "RNA genes" that code for various kinds of RNA that doesn't wind up being translated into proteins.

Such non-coding RNA can be further classified into things like ribosomal RNA, microRNA, small interfering RNA, and "long non-coding RNA".

This last, known as "long ncRNA" for short, is especially intriguing, because some studies have shown that there may be roughly four times as much of it (in base units) as there is of messenger RNA that is ultimately translated into proteins.

Even though a lot of these long ncRNAs are routinely found floating around inside cells, we're still in the dark about what, if anything, they actually do. But some recent research has revealed a little more about some long ncRNAs:

Early-stage Gene Transcription Creates Access To DNA (10/6/08)

Previously thought to be inert carriers of the genetic instructions from DNA, so-called non-coding RNAs turn out to reveal a novel mechanism for creating access to DNA required by transcriptional activation proteins for successful gene expression, according to Boston College Biology Professor Charles Hoffman, a co-author of the study with researchers from two Japanese universities. ...

Hoffman and his colleagues examined how the yeast cell senses its cellular environment and makes decisions about whether or not to express a gene, in this case fbp1, which encodes an enzyme. What they found was a preliminary transcription phase with a flurry of switches flicked "on" and then "off" as seen by the synthesis of non-coding RNA before the final "on" switch is tripped.

The non-coding RNAs initiate over one thousand base pairs of nucleotides along the DNA away from the known start site for this gene. The group discovered that the process of transcribing non-coding RNAs is required for the eventual production of the protein-encoding RNA. The transient synthesis of these non-coding RNAs serves to unfurl the tightly wound DNA, essentially loosening the structure to allow for gene expression. [Emphasis added.]

And here's the research article, with some of the abstract, providing a somewhat more precise description of what's going on:

Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs

Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1⁺ locus during transcriptional activation. The chromatin at fbp1⁺ is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1⁺. This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1⁺ transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.

To expand on that just a bit, recall that chromatin is the form in which DNA is actually stored for safe keeping. It consists of the double-stranded DNA molecules wrapped around many protein complexes called nucleosomes. Before any stretch of DNA can actually be transcribed into messenger RNA, the DNA has to be unwound from the nucleosomes. The present research has determined that some long ncRNA takes part in this unwinding process.

Tags: non-coding RNA, junk DNA, gene expression

Alternative splicing

Not so very long ago it used to be that molecular biologists thought that for every protein in the body there was a specific gene, and every gene contained the instructions for making just one protein. Then, when the human genome was completely mapped several years ago, it was found, to everyone's embarrassment, that there were a lot fewer than 25,000 different genes in the genome. This is in a genome of 3.12 billion base pairs. And the human genome is far from the largest. Ordinary corn has 5 billion base pairs and 50,000 genes. The trumpet lily plant (Lilium longiflorum) has 90 bilion base pairs in its genome, and the marbled lungfish (Protopterus aethiopicus) has 139 billion – but apparently nobody has had the patience to sit down and count their actual genes. (Reference; see also here.)

Anyhow, it's estimated that humans use at least 100,000 different proteins, maybe a lot more, so the point is that some genes must be capable of coding for a lot more than just one protein. It's now understood that this is accomplished by the process known as alternative splicing. As you know, genes are not simple, uninterrupted sequences of base pairs. They have within them several subsequences known as exons and introns. In a nutshell, the exons are eventually transcribed into messenger RNA, while the introns are discarded.

Except there's a little more to it than that. In order to produce different proteins, it's necessary to select a subset of exons to code for each particular protein. So how does this actually happen? Some new research has figured this out in one specific case:

RNA Map Provides First Comprehensive Understanding Of Alternative Splicing

It's biology's version of the director's cut. In much the same way that numerous films could be stitched together from a single reel of raw footage, a molecular process called alternative splicing enables a single gene to produce multiple proteins. Now a new RNA map, created by a team of researchers at Rockefeller University and the Howard Hughes Medical Institute and announced in the journal Nature, shows for the first time how the specific location of short snippets of RNA affects the way that alternative splicing is controlled in the brain.

Though scientists have begun to appreciate how alternative splicing adds a layer of complexity to brain processes that enable us to think and learn, exactly how alternative splicing is regulated during these processes -- and in some cases is uncontrolled (or dysregulated) to cause disease -- has remained elusive. The map provides the first comprehensive understanding of how alternative splicing works throughout the genome. The results have implications for a better understanding of such brain functions as learning and memory, neurological diseases and cancer biology.

To make a long story short, there is a brain protein called Nova that was known to be capable of binding to 50 different sequences of RNA. The study found that there were actually 30 different exons which contained those sequences, and whether or not a given sequence had been bound by Nova could cause the exon to be either included or excluded (depending on circumstances) from a final transcript.

This is of more than just theoretical interest. Errors in the transcription process can cause a variety of disease conditions:

By offering a global understanding of how alternative splicing works across the genome, the map has implications for the treatment of a growing list of human neurologic diseases in which RNA regulation, and particularly RNA splicing, has been implicated as the primary cause, including certain types of cancer and a number of brain and muscle disorders.

"Given that the complexity of the brain is orders and orders of magnitude more complex than the number of genes we have, one of the intriguing things about alternative splicing is that a relatively small number of regulatory splicing factors acting in concert on a single transcript can potentially generate a large number of different protein variants," says Darnell.

"There is a converging set of observations indicating that as neurologic diseases are better understood, alternative splicing is going to play an important role in generation of disease and therefore an important role in normal generation of cognitive function," he adds. "Our new work lays out an approach to developing a global understanding of how alternative splicing is regulated by one disease-associated protein, Nova, offering a route by which scientists may now be able to approach a number of diseases with a fresh start."

It's interesting, also, that this process is being observed in the brain. Because, as Antonio Damasio has just predicted for New Scientist as one of the most likely discoveries of the next 50 years, we should learn how relatively few genes can create such complexity in the brain:

Most of what I regard as exciting in recent neuroscience has concentrated on two broad areas: molecular neurobiology and an understanding of the systems related to cognition and behavior. The future will no doubt promote advances in those two areas. On the molecular side, it will be possible to know how so few genes (relatively speaking) create so much complexity in the human brain.

It would be a good guess that the use of alternative splicing is pretty common in brain tissue.

Update: And in fact, I wrote about this very topic a year ago: RNA splicing occurs in nerve-cell dendrites. The interesting thing is that in most cells, splicing is known to occur only in the nucleus. In neurons, however, it occurs in dendrites, the part of a neuron to which other neurons form connections.

Tags: medicine, biology, alternative splicing