RNA's Outfits

These days, RNA basks in an endless stream of reports about important new roles for little snippets of the nucleic acid, such as regulating the time and location of gene expression. This RNA renaissance has elevated the molecule far above its textbook role as middleman between DNA and proteins. But among the many accounts of RNA's exciting new biological roles, little, if any, note has been taken of the extraordinary diversity of ways in which the four nucleosides in this genetic molecule—adenosine, cytidine, guanosine, and uridine—are chemically modified.

RNA has an extraordinary wardrobe of chemical costumes. Some 120 different RNA modifications have been found in animal and bacterial cells, nearly 100 of which exist in humans. These modifications range from the fancy—installation of exotic sugars and other ring structures—to the relatively mundane, such as addition of methyl groups to RNA's bases or ribose backbone.

Genomic DNA, in contrast, is modified in only a handful of ways. Even so, DNA's comparatively pedestrian modifications have launched an entirely new branch of chemical biology called epigenetics.

Meanwhile, researchers have pinpointed a functional role for just a few of RNA's array of modifications. The biological role of "the vast majority of RNA modifications remains unclear," says Tao Pan, a biochemist at the University of Chicago. Still, researchers are slowly navigating the delicate complexity of these modified nucleosides in an attempt to figure out why RNA has so many chemical costumes.

One of the most modified of all RNAs is in the ribosome, the RNA-protein machine that translates messenger RNA (mRNA) into protein. The ribosome has approximately 200 modified nucleosides, which is about 3% of the RNA component of the macromolecule. Roughly 10% of the nucleosides in transfer RNA (tRNA), the biomolecules that recognize amino acids and carry them to the ribosome to be added to a growing protein chain, are modified. Regulatory RNAs—including the currently en vogue small nucleolar RNAs, microRNAs, and Piwi RNAs—also boast unusual modifications.

Although myriad RNA modifications exist, the two most common types are the posttranslational isomerization of uridine into pseudouridine and the methylation of RNA's ribose backbone at the 2′ position (creating 2′-O-methyl derivatives of adenosine, cytidine, guanosine, and uridine).

One of the biggest challenges in figuring out what biological role such modified nucleosides might play is that, in most cases, when a given modification is removed—by knocking out the gene that codes for the enzyme that performs the modification—nothing obviously different happens, explains Henri Grosjean, an enzymologist at Paris-Sud University, in France. Yet when two or more enzymes are removed, cells die. These observations have led researchers in the field to conclude that RNA modifications work in synergy to achieve their goals.

"Say you sit on a chair with four legs," Grosjean says. "Usually, the experiment people do is to remove just one leg. And of course you can still sit on a chair with three legs, even if it is not secure. But take away a second leg out of four, and it is no more a chair you can sit on."

The subtle role that RNA modifications play is perhaps exemplified by an experiment where microbiologists cultivate bacterial cells in a bioreactor. At the beginning of the experiment, they add an equal number of normal bacteria and mutant bacteria that have been engineered to lack an enzyme for making a particular RNA modification but otherwise appear healthy. This microbial cocktail is left to its own devices. After 1,000 divisions, microbiologists find only the normal bacteria in the reactor. Those bacteria lacking the modification enzyme fail to compete with the normal cells and disappear, Grosjean says. The take-home message, Pan says, is that "modified bases are probably useful for adaptation purposes, such as when cells are stressed, or in an environment where they have to have evolutionary advantage."

One instance where cells have used RNA modifications to achieve such an evolutionary advantage is in bacterial resistance, says Janusz M. Bujnicki, a bioinformatician at the International Institute of Molecular & Cell Biology, in Warsaw. For example, pathogenic bacteria, such as streptococci or staphylococci, often methylate bases on their ribosomes to sidestep effects of erythromycin and other macrolide antibiotics that target the ribosome. Bujnicki is using in silico and experimental methods to screen for inhibitors of the enzymes that install these methyl groups.

At the moment, there are just two situations in which researchers suspect RNA modifications are absolutely essential, both of which involve tRNA. Modified bases may expand the chemical portfolio of the four bases in tRNA, allowing a given tRNA to selectively recognize the individual amino acid that it is required to shuttle, says Mark Helm, a professor of pharmacy at the University of Heidelberg, in Germany. Those modifications may also help the tRNA find the right drop-off point for the amino acid on the mRNA that codes for the growing protein chain, he notes. Sections of tRNA that recognize amino acids and mRNA tend to be heavily modified, he adds. For example, the modification of a particular uridine to pseudouridine is "important for the accuracy of how tRNA recognizes the mRNA" so that the correct amino acid is delivered, Helm explains.

RNA modifications also seem to be essential where they serve to stabilize the three-dimensional structure of tRNA molecules. For example, the tRNA that transfers lysine to a growing peptide chain in human mitochondria requires a 1-methyladenosine modification to maintain its 3-D structure, Helm says. Unlike proteins, which have 20 amino acids whose physicochemical properties allow proteins to typically adopt a unique 3-D conformation, RNA "has only four bases to choose from," Helm explains. RNA structures can have many low-energy conformation options for their overall topology—yet they need to be stable enough to fulfill their duty as catalysts (in the ribosome's case) or carriers (in tRNAs' case). Modifications can help establish such a stable 3-D structure, Pan says.

Modified bases may also be playing an indirect role in the biology of a rare disease called MERRF (myoclonic epilepsy with ragged red fibers). Patients with this condition show a variety of muscular wasting symptoms, Helm says. Affected individuals lack a so-called wobble modification in a tRNA specific for lysine, so the tRNA cannot recognize where to drop off the amino acid on mRNA. "In fact, the missing wobble modification is a secondary effect of an inherited mutation within the core of the tRNA molecule," he says.

Even as researchers scramble to figure out the biological role of RNA modifications, others in the field are looking to harness these modified bases as biomarkers for diseases. Bernd Kammerer, a medical researcher at University Hospital TÜbingen, in Germany, and others have found that breast cancer patients excrete more of certain modified RNA bases in their urine than healthy women do; methylated nucleosides such as 1-methylinosine, sometimes found in tRNA, and pseudouridine seem to be particularly abundant. Kammerer believes that the reason for the extra modified bases is that RNA turnover seems to be impaired in cancer because phosphorylase enzymes that control such recycling are down-regulated in cancer cells. With less recycling, the modified bases must be excreted some other way and thus come out in urine. Other researchers have also looked at using the rare nucleoside 5′-deoxycytidine as a potential urinary biomarker forhead and neck cancer, he notes.

Modified RNA bases may be ubiquitous, but researchers in the field say that those outside the field are barely aware of such modifications' existence, much less their reach, according to Grosjean. He says this is partly because many biochemistry textbooks don't mention RNA modifications. But more important, he says, current lab practices make it easy to miss their presence. "If you don't look specifically for modifications, you won't discover them."

When researchers want to sequence RNA, most of them turn to the polymerase chain reaction (PCR), which produces a large number of the DNA molecule that has complementary bases to the RNA being sequenced. For 90% of modifications, Grosjean says, the polymerase that translates the original RNA sequence to DNA can't distinguish between a modified base and its parent base. So researchers may be getting RNA sequences through PCR, but they are losing modification information in the process.

Only RNA modifications that do not allow Watson-Crick base pairing—such as 1-methylguanosine or 1-methyladenosine—interfere with PCR enzymes and bring transcription to a halt. Because these modifications are particularly rare, most researchers encountering quirky PCR results just figure there was a break in the original RNA, Grosjean says. Compare this with "20 years ago, when people directly sequenced RNA" and modified bases were more on a biochemist's radar, he adds.

Another reason modified bases may not be widely appreciated is that studying them can be "a pain," Pan says. For example, researchers haven't yet managed to sequence every tRNA in humans. Studying modified bases in a high-throughput manner remains challenging not only because many of the RNA molecules in which they're found are transient, such as mRNA, but also because so many different modifications often exist in the same molecule, such as tRNA. That many modifications are enzymatically reversible also poses problems. Some researchers wonder whether bases are transiently modified as part of, say, some regulatory process or whether modifications are long-term decorations, Pan says. To sort this out, he's developed a ligation-based method that can detect some of the dynamics of RNA modifications.

Further complicating things is that it remains hard to quantify the presence of different modifications in a cell. Many researchers in the field say figuring out the exact type and quantity of modified RNA bases in cells would be a major step forward. Researchers, including Thomas Carell, a chemist at Ludwig Maximilians University, in Munich, are applying mass spectrometry and proteomic techniques to measure quantities of different modified RNA bases in cells.

Many researchers in the RNA-modification community are also trying to untangle the complicated enzyme pathways that lead to some of the more exotic chemical decorations. Tsutomu Suzuki of the University of Tokyo has married genomewide screening with mass spectrometry to find buried genes involved in making RNA modifications. Indeed, over the past decade, researchers have pinpointed more than 100 enzymes involved in RNA modifications, revealing a dichotomy between how bacteria modify their RNA and how higher organisms do so. In bacteria, enzymes that make the RNA modifications generally operate as separate entities, whereas in eukaryotes such as humans, the multiprotein complexes usually introduce the modifications. Those seeking to understand the enzymology of RNA modifications have been bolstered by recent X-ray crystallography structures of components of a pseudouridine-forming enzyme complex, for example.

With many mysteries remaining—such as the modifications for which no enzyme has been found and the scarcity of knowledge about human RNA modifications—the field is wide open for researchers seeking to know why these curious bases lurk in our cells.