Textbook Explanation Of mRNA Translation May Need Rethinking

Our understanding of how messenger RNAs are translated into proteins is challenged by new research published today in the Open Access journal Journal of Biology. The study suggests that EF-G, the GTPase that facilitates tRNA translocation in bacteria, enters the ribosome bound to a different guanine nucleotide than previously thought – GDP, not GTP. The ribosome itself then seems to act as the guanine-nucleotide exchange factor, not some as-yet-unidentified factor as previously assumed. This finding questions the prevailing model for RNA translocation.

According to the textbook model EF-G provides the energy needed for the translocation phase of translation by bringing GTP into the ribosome where GTP is subsequently hydrolysed into GDP.

Andrei Zavialov, Vasili Hauryliuk and Måns Ehrenberg from Uppsala University in Sweden first performed an important purification step ensuring that their GTP was not contaminated by GDP (and vice versa), as had been the case with previous studies using these purified components. They next measured the affinity of EF-G for GTP and GDP. Their results strongly suggest that EF-G is bound to GDP in the cytoplasm and that it binds to the pre-translocation complex - composed of the ribosome, tRNA and mRNA strand – as a EF-G-GDP complex. The ribosome itself then seems to act as a GTP exchange factor that swaps GDP for GTP, which results in a modification in the structure of the ribosome. This triggers partial translocation of the mRNA, which is completed after GTP hydrolysis. "Our results suggest that the ribosome plays a previously unidentified dual role of both guanine-nucleotide exchange factor and GTPase-activating protein" explain the authors. EF-G then detaches from the ribosome in its GDP-bound form, ready to be used again by another ribosome.

These findings differ radically from all previous models and as such may represent a considerable step forward in our understanding of translocation, a fundamental mechanism in protein synthesis and gene expression. RNA translation is a highly conserved mechanism and these results using a bacterial system are likely to be applicable to higher organisms as well. This should spur more research in the field to confirm or disprove the findings and give us a clearer picture of RNA translation. In particular, the present clarification of the translocation process at the biochemical level may allow a deeper understanding of how relative movements of the ribosomal subunits can accomplish thousands of translocation events without frame-shifting or loss of tRNA-bound nascent protein chains during peptide elongation.

BioMed Central

Molecular Steps Involved In The Creation Of Gene-Silencing MicroRNAs Identified

First discovered only a few brief years ago, microRNAs are small, remarkably powerful molecules that appear to play a pivotal role in gene silencing, one of the body's main strategies for regulating its genome. A scant 22 nucleotides in length, miRNAs appear to work by binding to and somehow interfering with messenger RNA, itself responsible for translating genes into proteins.

But how do miRNAs arise? And what can we learn about their biological function from their origins? In a study published last year in Nature, researchers at The Wistar Institute identified a two-protein complex, called the microprocessor complex, which controls the earliest steps in the creation of miRNAs in the cell nucleus.

Now, in a new study published online by Nature today, the Wistar group has identified a three-protein complex that picks up the process in the cell cytoplasm and carries it through to the maturation of the finished miRNAs.

Taken together, the two Nature studies trace the generation of miRNAs from the genes that give rise to long primary RNA molecules through to the mature miRNAs that target messenger RNA. Significantly, the research also shows that the finished miRNAs are associated with a protein called Argonaute 2, known to be involved in inactivating messenger RNA.

"In this study, we were able to link processing of the miRNAs directly through to the molecules responsible for silencing genes," says Ramin Shiekhattar, Ph.D., an associate professor at Wistar and senior author on both Nature studies. "The miRNAs provide specificity for those molecules, which do the actual work of gene silencing."

Intriguingly, the research also links the process of creating miRNAs with aspects of the HIV life cycle and with tumor suppression. The study identifies three proteins that work together in the cytoplasm to create finished miRNAs. Individually, each of the proteins was known previously, but their joint role in producing miRNAs is newly delineated here. Equally as important, however, is the fact that while two of the proteins had been associated with miRNAs in earlier work, the third protein, TRBP, had not been. And TRBP is clearly a protein of interest to scientists.

"TRBP was first observed as a protein that binds to HIV during transcription of the virus," says Shiekhattar. "The tantalizing implication is that the RNA interference pathway may play a significant role in HIV replication. TRBP has also been identified as a tumor suppressor, which suggests still other connections to be explored."

The Wistar Institute

Study Shows How Sleep Improves Memory

A good night's sleep triggers changes in the brain that help to improve memory, according to a new study led by researchers at Beth Israel Deaconess Medical Center (BIDMC).

These findings, reported in the June 30, 2005, issue of the journal Neuroscience and currently published on-line, might help to explain why children – infants, in particular – require much more sleep than adults, and also suggest a role for sleep in the rehabilitation of stroke patients and other individuals who have suffered brain injuries.

"In this new research, by using functional magnetic resonance imaging (fMRI), we can actually see which parts of the brain are active and which are inactive while subjects are being tested, enabling us to better understand the role of sleep to memory and learning."

New memories are formed within the brain when a person engages with information to be learned (for example, memorizing a list of words or mastering a piano concerto). However, these memories are initially quite vulnerable; in order to "stick" they must be solidified and improved. This process of "memory consolidation" occurs when connections between brain cells as well as between different brain regions are strengthened, and for many years was believed to develop merely as a passage of time. More recently, however, it has been demonstrated that time spent asleep also plays a key role in preserving memory.

In this new study, twelve healthy, college-aged individuals were taught a sequence of skilled finger movements, similar to playing a piano scale. After a 12- hour period of either wake or sleep, respectively, the subjects were tested on their ability to recall these finger movements while an MRI measured the activity of their brain.

According to Walker, who is also an Assistant Professor of Psychiatry at Harvard Medical School, the MRI results showed that while some areas of the brain were distinctly more active after a period of sleep, other areas were noticeably less active. But together, the changes brought about by sleep resulted in improvements in the subjects' motor skill performance.

"The cerebellum, which functions as one of the brain's motor centers controlling speed and accuracy, was clearly more active when the subjects had had a night of sleep," he explains. At the same time, the MRIs showed reduced activity in the brain's limbic system, the region that controls for emotions, such as stress and anxiety.

"The MRI scans are showing us that brain regions shift dramatically during sleep," says Walker. "When you're asleep, it seems as though you are shifting memory to more efficient storage regions within the brain. Consequently, when you awaken, memory tasks can be performed both more quickly and accurately and with less stress and anxiety."

This new research may explain why children and teenagers need more sleep than adults and, in particular, why infants sleep almost round the clock.

"Sleep appears to play a key role in human development," says Walker. "At 12 months of age, infants are in an almost constant state of motor skill learning, coordinating their limbs and digits in a variety of routines. They have an immense amount of new material to consolidate and, consequently, this intensive period of learning may demand a great deal of sleep."

The new findings may also prove to be important to patients who have suffered brain injuries, for example, stroke patients, who have to re-learn language, limb control, etc.

"Perhaps sleep will prove to be another critical factor in a stroke patient's rehabilitation," he notes, adding that in the future he and his colleagues plan to examine sleep disorders and memory disorders to determine if there is a reciprocal relationship between the two.

Beth Israel Deaconess Medical Center

Exercise and IGF-1 Act Synergistically to Prolong Survival in Experimental ALS

NEW YORK (Reuters Health) - In a mouse model of amyotrophic lateral sclerosis (ALS), running on an exercise wheel further boosts the increased survival associated with insulin-like growth factor-1 (IGF-1) gene therapy, investigators report.

Dr. Fred H. Gage, at the Salk Institute for Biological Studies in La Jolla, California, previously showed that IGF-1 treatment using an adeno-associated virus (AAV) vector prolongs survival in transgenic mice that overexpress superoxide dismutase-1 (SOD1), a model of ALS.

For their current report, published in the Annals of Neurology for May, Dr. Gage's group placed SOD1 mice with a running wheel, beginning before symptom onset (age 40 days) or after onset (90 days) and compared outcomes with those of sedentary mice.

Early exposure to the wheel for 2 hours/day increased median survival by 7 days (p < 0.0005) over the median survival of 122.5 days seen in nonrunning animals. Exposure for 6 and 12 hours increased median survival to 163 days and 147 days, respectively.

In a separate set of experiments, the authors showed that AAV-IGF-1 treatment increased median survival compared with controls (148 days survival versus 119 days in untreated mice). Early running without AAV-IGF-1 treatment led to a similar prolongation of survival (to 141 days).

Treatment at day 90 with AAV-IGF-1 along with early running increased survival most of all (83-day increase, to 202 days, p < 0.0001). Postponing wheel exercise to 90 days of age led to a smaller benefit in AAV-IGF-1-treated animals (survival to 156 days).

Whereas nonrunning mice exhibited a 50% decrease in the number of motor neurons in the lumbar spinal cord at 110 days compared with wild-type mice, early running, AAV-IGF-1 treatment or combined treatment was associated with no significant change in motor neuron counts, the investigators report. Both modalities decreased astrogliosis compared with sedentary, nontreated animals.

Early running, but not AAV-IGF-1 treatment, was also associated with significantly increased levels of the antiapoptotic genes Bcl-xL and Bcl-2 in the lumbar spinal cord, in comparison with wild-type animals.

None of the animals showed any significant changes in SOD1 expression, "suggesting that the therapeutic effect of running and/or IGF-1 were acting through signaling pathways versus downregulating mutant SOD1 protein," Dr. Gage's group suggests.

Summing up, the team writes, "we have shown that exercise promotes motor neuron survival, attenuates astrogliosis, improves motor function and extends survival."

They propose that "exercise in conjunction with IGF-1 gene therapy may provide the most efficacious treatment for ALS to date."

Ann Neurol 2005;57:649-655.

RNA Silencing of SOD1 Preserves Strength in Mouse Model of ALS

NEW YORK (Reuters Health) - Intramuscular injections of a viral vector encoding short interfering RNA (siRNA) directed against superoxide dismutase (SOD1) delays the loss of grip strength in a mouse model of amyotrophic lateral sclerosis (ALS), investigators report in the May Annals of Neurology.

Mutations in SOD1 cause one form of dominantly inherited ALS in humans, senior investigator Dr. Don W. Cleveland and his colleagues note. Transgenic mice that overexpress mutant SOD1 develop symptoms that resemble ALS in humans.

To test the efficacy of treatment with siRNA, Dr. Cleveland, at the University of California, San Diego, in La Jolla and his team designed an adeno-associated virus (AAV-2) that carried green fluorescent protein (GFP) and siRNA directed against SOD1 or a 2 base-pair missense control. They then injected the lower part of the hind limb on one side of SOD1 mutant mice with one of the two AAV-2 viruses and tested the animals weekly with a grip strength meter.

GFP was found in the spinal cord of all the injected animals, demonstrating retrograde transport of the virus, the authors note, and motor neurons expressing GFP had lower levels of SOD1.

Only the limbs of the mice that were treated with the SOD1-directed siRNA showed "remarkable preservation of grip strength," compared with the non-injected control limb.

"We anticipate that reduction in mutant SOD1 by AAV-2-mediated delivery of siRNA after peripheral injection may be an effective therapy for SOD1 familial ALS patients," Dr. Cleveland and his associates maintain.

Ann Neurol 2005;57:773-776.