Short RNAs to the Rescue

Ever since scientists realized, just over a decade ago, that exposing cells to short snippets of RNA could affect the activity of matching genes, they have dreamed if harnessing this RNA interference, or RNAi, to fight diseases. In the past week, two groups have announced progress toward that goal, treating chimpanzees with hepatitis C and mice with lung cancer.

RNAi, which rapidly earned a 2006 Nobel Prize, is just one facet of the many ways in which short RNAs regulate gene activity. Researchers have since found numerous types of naturally occurring short RNA that play important roles in development, stem cells, cancer, and other biological processes. These RNA-based mechanisms could seriously revise the emerging understanding of how cellular processes are controlled.

Over the same period, manipulating genetic activity with short RNAs has become an essential tool in biology labs. Cells process various forms of short RNA, such as short-hairpin RNA (shRNA) and small interfering RNA (siRNA) into RNA-protein complexes that reduce (usually) how much protein is made from a messenger RNA that include a complementary (or nearly complementary) sequence.

This technique gives researchers a quick way to learn about what a particular gene does, at least in culture dishes, sidestepping the laborious creation and breeding of genetically-modified critters. (Or if they do put in the time, they can insert genes that allow them to controllably trigger RNAi to knock down a gene only in particular cells or after it has completed an indispensible task in helping an organism to grow.)

But affecting genetic regulation in patients faces the challenges of "delivery" that are well-known in the pharmaceutical industry: To have a beneficial effect, the short RNA must survive in the body, get inside the right cells in large quantities, and not cause too many other effects in other cells. The New York Academy of Sciences has a regular series on the challenges of using RNA for treatment, and I covered one very interesting meeting in 2008.

Molecular survival is the first challenge. Researchers have developed various chemical modifications that help RNA (or a lookalikes) withstand assaults by enzymes that degrade rogue nucleic acids. Santaris, for example, which helped in the hepatitis project, has developed proprietary modifications it calls "locked nucleic acids," or LNA. Other researchers and companies are exploring similar techniques.

Getting the protected RNA to the right tissue is another challenge. Foreign chemicals are naturally cycled to the liver for processing, so it's fairly easy to target this organ. For this reason, the hepatitis results don't really prove that the technique is useful for other tissues. The Santaris release also neglects to mention any publication associated with the research.

The mouse lung cancer result appears in Oncogene. The lead Yale researcher, Frank Slack, regularly studies short RNAs in the worm C. elegans, as I described in a recent report from the New York Academy of Sciences. In this work, he teamed with Mirna Therapeutics, which aims to use the short-RNA-delivery vehicle to replace naturally occurring microRNA that are depleted in cancer, like the let-7 they used for this study. The mouse cancers did not disappear, but they regressed to about a third of their previous size, according to the release. Mirna says that since they are replacing natural microRNAs, their technique shouldn't induce many side effects in other tissues.

A further risk for small-RNA delivery is immune responses. The field of gene therapy is only now recovering from the 1998 death of Jesse Gelsinger in what looks like a massive immune response to the virus used to insert new genes in his cells. Although the short-RNA response will be different, some cellular systems are primed to respond to the foreign nucleic acids brought in by viruses.

It's likely that there will be many twists and turns along the way, and I haven't solicited expert opinions on these studies, but they seem to be intriguing steps toward the goal of using RNA not just to study biology, but to change people's lives.

Alternative Splicing

In 1977, researchers were surprised to learn that the protein-coding sequence of messenger RNA doesn't arise from a continuous section of DNA.

Instead, work that earned Phil Sharp and Richard Roberts a Nobel in 1993 found that the as-transcribed pre-mRNA includes sections called introns that are then cut out of the sequence while the remaining exons are spliced back together (the words can apply to either DNA or RNA).

The final protein-coding section is straddled on both ends, called 5' and 3', by untranslated regions (UTRs). These noncoding regions are also transcribed from the DNA, but aren't usually described as exons. But their sequence still matters: out in the cell, the 3' UTR is a favorite target for complementary microRNAs that affect the stability or translation of the messenger RNA.

Additional processing steps in the nucleus add to the spliced-together RNA a trademark chemical cap at its 5' end and a tail of repeated adenylenes at its 3' end. Both the cap and the polyadenylated tail are important for the later translation of the mature mRNA at ribosomes, once it has been exported from the nucleus.

A further wrinkle was the realization that the splicing can happen in different ways, as illustrated in the figure (from Wikipedia), which connects by blue lines the pieces that can be neighbors in the final RNA. The multiplicity of possible proteins resulting from this alternative splicing significantly increases the number of protein products available from a given stretch of DNA. Most human proteins occur in more than one splicing arrangement, called an isoform.

The splicing is done by a large complex of RNA and proteins called the spliceosome. The choice of isoform depends in part on special RNA sequences, either within an exon or an intron, that bind proteins that promote or inhibit splicing at a particular point. This binding is sensitive to sequence changes that don't change the coded amino acid and are therefore called "synonymous." Because of splicing, these changes aren't always synonymous: they change the final protein.

In addition, the relative amounts of the alternatively spliced isoforms can change, for example, during development of an organism in different tissues, notably the brain. The regulation of this process provides yet another tool for controlling gene expression, but scientists are still clarifying what determines the splice configuration.

To get a global view of alternative splicing, Chris Burge of MIT, at a conference that I covered last year, described a technique called mRNA-seq that preferentially sequences short RNA segments that contain the polyadenylated tail, and are therefore proper mRNA candidates for later translation. (This eliminates the confusing background of transcribed RNA that is useless or acts in other ways.) Using this technique, he and his colleagues identified more than 10,000 sequences that coded for multiple isoforms. Of these, Burge estimated that more than 2/3 were present in different amounts in different tissues, so they different forms seem likely to be doing important things.

Burge also found a surprising connection between the processes that add the polyadenylene tail and those that do splicing. The former process occurs at the end of transcription, but it looks as if the RNA is already being handed off to the splicing machinery before transcription is finished.

Although it is still poorly understood, alternative splicing is an important and widespread mechanisms for regulating genes, as well as for getting multiple proteins out of a single region of DNA.

Short RNAs in Stress and Longevity

My latest eBriefing for the New York Academy of Sciences covers a day-long May 12 meeting on "Short RNAs in Stress and Longevity." (The web publication was delayed by redesign and reorganization disruptions at the academy).

The sponsor, the Non-coding RNA Biology Discussion Group, used to call itself the RNA Interference Discussion Group. Its new name more accurately reflects the diverse regulatory and other roles of short RNAs. Many of these roles are far from being completely understood, and this meeting touched on several of them.

The combination of stress and longevity may seem like an odd pairing. But to the extent that many organisms have a built-in, switchable longevity program, stresses like starvation, which make living longer more attractive than reproducing, can activate it. The stress response is an entire field of its own, and stresses like heat shock are well known to produce major shifts in gene expression, inducing production of proteins like chaperones that help cells cope.

Frank Slack of Yale University and Ramanjulu Sunkar of Oklahoma State University explored two fields where researchers have extensively studied gene regulation by traditional protein-based mechanisms. For longevity in the worm C. elegans and for stress responses in plants, respectively, they saw the effects of naturally-occurring short RNAs (microRNAs and their plant relatives), and changed these responses by manipulating the short RNA levels. As in other fields, these studies are revealing a critical layer of gene regulation that has been overlooked until quite recently.

Germano Cecere works in the lab of Alla Grishok of Columbia University, who had found that short RNAs can regulate not just messenger RNA translation and degradation, but also its initial transcription from DNA. To find new examples, Cecere searched for short RNAs that are involved with chromatin remodeling, and identified many that play a role in stress and longevity.

Cells under stress often develop specialized complexes of proteins and RNA, known as stress granules, which may host some of the regulatory reactions or store low-priority messenger RNA. Anthony Leung of Phil Sharp's lab at MIT described how a particular polymer known for its role in DNA processes might act as a scaffold for these granules or even help regulate their activity.

Irina Groisman of the André Lwoff Institute dissected protein complexes that associate with the poly-adenylated tails found in mature messenger RNA. These complexes enforce the tradeoff between cellular senescence, which is related to longevity, and cancer.

Beyond the regulatory sequences that typically contain twenty-something bases, larger RNA can also process information on its own. Evgeny Nudler of NYU, who had identified riboswitches that respond to metabolites, described a different 600-nucleotide-long RNA that is the temperature-sensing element in the heat-shock response. This RNA sensor binds with the translational elongation factor eEF1A (which can also interact with non-heat stresses) to generate the response.

As this breathless summary hints, it's a challenge to combine such disparate topics into a coherent writeup. In this case, with just six talks, I gave up on aligning the talks into common themes and simply summarized each one separately. This diversity shows how wide open the field of RNA remains, going far beyond its traditional functions as messenger RNA, transfer RNA, and ribosomal RNA.

Ribosomes

The 2009 Nobel Prize in Chemistry was awarded to Venkatraman Ramakrishnan, Thomas Steitz, and Ada Yonath for their elucidation of the structure of ribosomes and how that structure promotes accurate translation of messenger RNA sequences into the amino acid sequences of proteins.

Ribosomes are the granddaddies of the ribonucleoprotein machines in the cell--big enough to be customarily granted organelle status even though they don't have a membrane. The bacterial version of the complex, for example consists of two large parts, denoted 30S and 50S to represent how fast they separate out of a suspension. Each of these subunits contains many proteins (20 and 33, respectively), together with large "ribosomal" RNA chains.

With the assistance of transfer RNA, ribosomes translate messenger RNA sequences (previously transcribed from DNA in the nucleus) into a corresponding amino-acid sequence or polypeptide, which will be folded and processed into a mature, functioning protein. In light of this critical and intricate task, it should probably not be surprising that the ribosome is very similar in widely different species. However, bacterial, archaeal, and eukaryotic ribosomes are rather different, and the differences in the ribosomal RNA were used by Carl Woese in the 1970s as the evidence for the highest-level classifying of life forms into these three broad domains.

Streptomycin, tetracycline, and about half of current antibiotics preferentially disrupt bacterial protein synthesis by attacking the bacteria-specific versions of the ribosome. The details of the ribosome structure can guide researchers who hope to develop new types of antibiotic molecules.

The three researchers and their collaborators all studied the structure of ribosomes by x-ray crystallography. This structure revealed specific details about how transfer RNA, with its individual matching amino acid cargo, nestles into the ribosome, and how the amino acid forms a covalent bond with the growing polypeptide. (The Nobel Committee notes that the charge-coupled devices that garnered this year's physics prize have made such studies much more productive.) Researchers have used these and other studies to clarify the entropy and energy that drives this synthesis.

A critical aspect of quality control in protein synthesis is the "proofreading" that ensures that the RNA sequence in the transfer RNA is indeed complementary to that of the messenger RNA. In 1974, John Hopfield (then at Princeton and Bell Labs) proposed that a multi-step ratchet that repeatedly checks the match while expending energy could be more selective than depending on the rather weak thermodynamic preference for a match. The increasingly refined structures revealed by the prize winners, together with other experiments, have confirmed how this proofreading works in the real ribosome, achieving an amazingly low error rate of about one error in 10,000 amino acids.

Soft Machines

Biological macromolecules, such as DNA, RNA and protein, spend much of their time in the company of other macromolecules, rather than floating freely on their own. The resulting molecular behavior in a cell resembles the choreographed handoff of a product between machines in an assembly line, rather than the indiscriminate reactions of molecules in a chemistry-lab beaker. The intriguing twist is that the "machines" are themselves collections of biological macromolecules that can also come and go.

Specialized cellular structures, known as organelles, are basic to any introduction to cell biology. But the usual suspects, such as the nucleus, mitochondria, chloroplasts, the endoplasmic reticulum and golgi apparatus, and various vacuoles, are all delineated by membranes. The lipid molecules of the membrane are also free to come and go, of course, but this construction makes it easy to define the organelles.

Other structures, often not called organelles because they lack a membrane, are just as important. The best known examples are ribosomes, which are large enough (about 0.02 microns) to have been noticed in electron micrographs some fifty years ago. They are complexes of specialized RNA with specific proteins, together known as ribonucleoprotein, or RNP. The ribosomes translate the sequence of bases in a messenger RNA strand into a corresponding amino-acid sequence in a growing protein.

Another structure, found in the nucleus, is the spliceosome. This critical RNP complex processes pre-RNA that is directly transcribed from the DNA, cutting out some sections and splicing the rest back together to form a proper protein-coding sequence. The spliceosome frequently splices in different sections of the pre-RNA to specify different variants of the protein, depending on cellular conditions. Once the strand is equipped with a cap and a tail on opposite ends, it is a messenger RNA ready for export from the nucleus.

Back outside the nucleus, the messenger RNA may encounter two other types of RNP structures, known as processing bodies and stress granules (the latter appear only in stressed cells). Messenger RNA can be temporary stored in either of these complexes, delaying its translation into protein by the ribosomes. Processing bodies can also permanently degrade the RNA preventing it from being translated. In ways that are still being explored, this degradation is associated with the RNA interference, in which short regulatory RNAs work with proteins to target messenger RNA strands that have a mostly complementary sequence.

Experiments show that molecules are constantly entering and leaving these complexes (at least some of them), unimpeded by any membrane, so there may be no clear line between them and the transient association of a few macromolecules. These dynamic association are similar to the shifting alliances of politicians in congress, some formal and restrictive like a political party, some more of an informal hallway conversation, but each contributing to the political process. Molecular complexes in the cell, though not always recognized, are equally critical to its function.

New Roles for RNA

The 2006 Nobel Prize in Physiology or Medicine went to Andrew Fire and Craig Mello for work that had been published only eight years earlier, in 1998. This rapid award reflected the enormous impact of their discovery that adding double-stranded RNA to cells can alter their expression of genes that were already transcribed into messenger RNA, an effect called RNA interference, or RNAi.

Manipulating the activity of specific genes, without needing to breed new organisms with altered DNA, has proven to be an extremely powerful laboratory tool for revealing the functions of those genes. Naturally, researchers also hope they can use it medically, pulling off the equivalent of gene therapy without the risks of messing with people's DNA. This is still a work in progress, because it's not trivial to deliver a largish RNA molecule intact to the right tissues and to avoid side effects.

In addition to the promise for manipulating gene expression for research or therapy, however, researchers have begun to realize that RNAi-like phenomena are critical to gene regulation in normal organisms (as well as being disrupted in some diseases). In this case, gene expression is altered, not by RNA added to the cells, but by naturally occurring RNA transcribed from parts of the genome that don't code for proteins.

I suspect that we will look back with smug superiority at the primitive era when people thought they might explain most gene expression changes to the action of protein transcription factors. For now, though, both the understanding and the terminology of the field are evolving rapidly.

It's important to understand that RNA doesn't act alone. Instead, it joins together in complexes with proteins. In particular, proteins from one family, the Argonautes, link up with one strand of the interfering RNA in a way that lets the combination recognize messenger RNA that contains a more-or-less complementary sequence. This lets the complex selectively target the messenger RNA for a particular protein, for example, out of all of the RNA in the cell.

Different protein-RNA complexes affect their target RNA in different ways. One type of complex slices up the matching messenger RNA. By decreasing the amount of the target RNA in the cell, this reduces the rate at which protein is translated from it. In a second important mechanism, a different RNA/protein complex directly slows (or speeds) translation of the target messenger RNA, without changing how much of it there is.

Researchers are only beginning to explore the networks of molecular interactions that involve these RNA-based processes, and how these molecular patterns change during normal cellular activities and in diseases like cancer.

Gene Regulation

Here's a question that every high-school biology student ought to ask: "if every cell in our body has the same DNA [which is almost true], why are they so different?" Understanding how different cells employ different parts of the common genome, turning some genes "on" and others "off," for example, is a long-standing problem in biology. As is typical for biology, there are many correct answers, some only now being elucidated.

Different cells have characteristic "signatures" of genetic activity. A neuron is a neuron because certain sets of genes, like those that specify proteins used in synapses, are active, while genes involved in synthesizing bile, for example, are not. The pattern also changes though the various stages of the cell cycle that lead to cell division. But such differences are only the tip of the gene-regulation iceberg.

A single-cell zygote develops into a complex multi-cellular organism like you or me through an intricate choreography that activates and represses genes in the appropriate cells at the appropriate times. This gene regulation is coordinated in time, as the activity of one gene triggers event that turn another on or off. It is also coordinated in space, as gene activity in one cell or region creates chemical signals that affect nearby cells. Perhaps it's not too surprising that the complex "network" of genes turning each other on and off can give rise to complicated patterns and time and space. But it is still close to miraculous that this complex process produces such complex creatures--with fingernails, bile ducts, and neural circuits that specialize in speech perception--with such astonishing robustness. (It fails frequently, but it's amazing that it works at all.)

Normally developing body cells change their patterns of expression irreversibly: once they have differentiated from a more general-purpose cell to a specialized cell, they don't go back again. This irreversibility is a somewhat surprising property of the interaction network, and presumably helps to assure proper development. Until recently, therefore, researchers looking to generate new cells for either research or therapeutic purposes had to isolate "stem cells" that had not yet gone through this irreversible differentiation. The most versatile such cells come from embryonic tissue only a few days after fertilization. These embryonic stem cells are still very important, but in the past few years researchers have successfully turned back the clock on fully differentiated skin cells, for example, apparently giving them all the versatility of embryonic stem cells.

Cancer cells also turn back the clock, activating genes that normally function only in early development. Some cancers, for example, turn on genes that induce nearby tissue to build blood vessels to supply the growing cancer. In ordinary cells, the genetic networks that enable this activity are present but inactive after they do their work in the growing embryo. The ability of cancer cells to re-activate these dormant cellular programs is a key to their success, but also gives researchers critical insight into the ways genes are regulated.

In future posts I will discuss various molecular mechanisms of gene regulation.