Genetic Discovery Will Revolutionize Understanding Of Gene Expression

Over the past decade, research in the field of epigenetics has revealed that chemically modified bases are abundant components of the human genome and has forced us to abandon the notion we've had since high school genetics that DNA consists of only four bases.

Over the past decade, research in the field of epigenetics has revealed that chemically modified bases are abundant components of the human genome and has forced us to abandon the notion we've had since high school genetics that DNA consists of only four bases. Now, researchers have made a discovery that once again forces us to rewrite our textbooks. This time, however, the findings pertain to RNA, which like DNA carries information about our genes and how they are expressed. The researchers have identified a novel base modification in RNA which they say will revolutionize our understanding of gene expression [Credit: © Attila Németh / Fotolia]

Now, researchers at Weill Cornell Medical College have made a discovery that once again forces us to rewrite our textbooks. This time, however, the findings pertain to RNA, which like DNA carries information about our genes and how they are expressed. The researchers have identified a novel base modification in RNA which they say will revolutionize our understanding of gene expression.

Their report, published in the journal Cell, shows that messenger RNA (mRNA), long thought to be a simple blueprint for protein production, is often chemically modified by addition of a methyl group to one of its bases, adenine. Although mRNA was thought to contain only four nucleobases, their discovery shows that a fifth base, N6-methyladenosine (m6A), pervades the transcriptome. The researchers found that up to 20 percent of human mRNA is routinely methylated. Over 5,000 different mRNA molecules contain m6A, which means that this modification is likely to have widespread effects on how genes are expressed.

"This finding rewrites fundamental concepts of the composition of mRNA because, for 50 years, no one thought mRNA contained internal modifications that control function," says the study's senior investigator, Dr. Samie R. Jaffrey, an associate professor of pharmacology at Weill Cornell Medical College.

"We know that DNA and proteins are routinely modified by chemical switches that have profound effects on their function in both health and disease. But biologists believed mRNA was simply an intermediate between DNA and protein," he says. "Now we know mRNA is much more complex, and defects in RNA methylation can lead to disease."

Indeed, as part of the study, the researchers demonstrated that the obesity risk gene, FTO (fat mass and obesity-associated), encodes an enzyme capable of reversing this modification, converting m6A residues in mRNA back to regular adenosine. Humans with FTO mutations have an overactive FTO enzyme, which results in low levels of m6A and causes abnormalities in food intake and metabolism that lead to obesity.

The researchers uncovered links between m6A and other diseases as well.

"We found that m6A is present in many mRNAs encoded by genes linked to human diseases, including cancer as well as several brain disorders, such as autism, Alzheimer's disease, and schizophrenia," says the study's lead investigator, Dr. Kate Meyer, a postdoctoral researcher in Dr. Jaffrey's laboratory. 

"Methylation in RNA is a reversible modification that appears to be a central step in a wide variety of biological pathways and physiological processes," she says.

The first time that m6A was detected in mRNA was in 1975, but at the time scientists were unsure whether this finding was a result of contamination by other RNA molecules, Dr. Jaffrey says. Over 90 percent of RNA is either transfer RNA (tRNA) or ribosomal RNA (rRNA), cellular workhorses that are routinely modified.

But Dr. Jaffrey says he has always been interested in the idea that mRNA may be modified - "DNA, proteins, other forms of RNA are modified, so why not mRNA?" he says - so he and investigators in his laboratory developed a technique to help them uncover methylation in mRNA taken from both mouse and human samples.

They used two different antibodies that recognize and bind to m6A in mRNA in order to selectively isolate the mRNAs that contain m6A. By subjecting these mRNAs to next-generation sequencing, they were able to identify the sequence of each individual mRNA they had isolated. Co-authors Dr. Christopher Mason and Dr. Olivier Elemento, assistant professors from the Department of Physiology and Biophysics and Computational Genomics in Computational Biomedicine at Weill Cornell Medical College, then developed computational algorithms to reveal the identity of each of these methylated mRNAs.

The Weill Cornell researchers don't know how the thousands of m6As they detected in humans work to control the function of mRNAs, but they do note that the m6As are located near "stop codons" in mRNA sequences. These areas signal the end of translation of the mRNA, suggesting that m6A might influence ribosomal function. "But we really don't know yet," says Dr. Mason, a co-lead investigator on the study. "It may allow other proteins to bind to mRNA, or subject these mRNAs to a whole new regulatory pathway. Our bioinformatics analyses are providing several hints about the possible impact of methylation on RNA function."

Indeed, in their study, the investigators have already found that m6A sites frequently occur in regions of mRNA that are highly conserved across several species of vertebrates. "This shows that m6A sites are not just important for humans, but rather are maintained under selection across hundreds of millions of years of evolution, and thus are likely of critical importance for all animals," Dr. Mason says.

"This is the first demonstration of an epitranscriptomic modification - alterations in RNA function that are not due to changes in the underlying sequence," he adds.

"These findings are very, very exciting, and amazing, really, when you consider that mRNA has been around for so long and that nobody realized, in all this time, that they were being regulated in this way," Dr. Jaffrey says. "It was right under our noses."

In addition to investigating how m6A regulates mRNAs within cells, the researchers are now focused on identifying the enzymes and pathways that control mRNA methylation.

Their study already demonstrates that FTO is capable of reversing adenosine methylation and suggests that it acts on a large proportion of cellular mRNA. "FTO mutations are estimated to occur in one billion people worldwide and are a leading cause of obesity and type 2 diabetes. Our studies link m6A levels in mRNA to these major health problems and identify for the first time the mRNAs which are potentially targeted by FTO," Dr. Meyer says.

The investigators are currently working to understand how defective regulation of m6A in patients with FTO mutations causes obesity and metabolic disorders, and they are also developing tests to rapidly identify compounds that inhibit FTO activity. These compounds are expected to inhibit the overactive FTO found in humans, potentially leading to novel therapeutics for diabetes and obesity.

Source: Medical News Today {May 19, 2012]

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Scientists identify defect in brain cell channel that may cause autism-like syndrome

Neuroscientists at Stanford University School of Medicine have homed in on potential differences in autistic people's brain cells by studying brainlike spheres grown in an elaborate process from skin cells. 

The scientists studied cells from patients with Timothy syndrome, a rare genetic condition that is associated with one of the most penetrant forms of autism: In other words, most people with the Timothy syndrome mutation have autism as a symptom, among other problems. 

Autism is a spectrum of developmental disorders of impaired social and verbal interaction. Currently, no medication exists to treat its underlying causes, according to the U.S. National Library of Medicine. Understanding what goes awry in autistic brain development could improve prospects for treating the condition. 

In this study, the scientists suggest that the autism in Timothy syndrome patients is caused by a gene mutation that makes calcium channels in neuron membranes defective, interfering with how those neurons communicate and develop. The flow of calcium into neurons enables them to fire, and the way that the calcium flow is regulated is a pivotal factor in how our brains function. 

The researchers also found brain cells grown from individuals with Timothy syndrome resulted in fewer of the kind of cells that connect both halves of the brain, as well as an overproduction of two of the brain's chemical messengers, dopamine and norepinephrine. Furthermore, they found they could reverse these effects by chemically blocking the faulty channels. 

Postdoctoral scholar Sergiu Pasca, MD, and Ricardo Dolmetsch, PhD, associate professor of neurobiology, led the study, which will be published online Nov. 27 in Nature Medicine. Dolmetsch, a biophysicist, redirected his research to study autism after his son was diagnosed with Timothy syndrome. It's unclear what leads to autism, but its incidence is increasing, he said. 

The gaps in our understanding of the causes of psychiatric disorders such as autism have made them difficult to treat. Perhaps the biggest obstacle to research into autism and other psychiatric and neurological diseases is that scientists can't get living brain cell samples from people with these conditions, for obvious reasons. Dolmetsch and his colleagues figured out a solution to this dilemma, using a novel approach involving what are known as induced pluripotent stem cells, or iPS cells. 

"We developed a way of taking skin cells from humans with Timothy syndrome and converting them into stem cells, then converting those stem cells into neurons," said Dolmetsch. 

The scientists grew these iPS cells as free-floating clumps in a nutrient-rich solution, later transferring the clumps to tissue culture plates. Here, some of them formed three-dimensional, brainlike spheres whose cells later migrated outward and matured into neurons. These neurons formed three distinct layers, a good first approximation of living tissue in the brain. By visualizing these neurons under a microscope and quantifying their gene expression, the scientists were able to characterize at the cellular level abnormalities that may be associated with autism. 

The neurons grown from Timothy-syndrome iPS cells showed larger-than-normal spikes in calcium levels, suggesting the calcium channels lost their ability to shut off. This set off dramatic changes in neuronal signaling, reconfiguring how genes were expressed. 

The cerebral cortex, the outer layer of the brain, has six distinct layers. In Timothy syndrome cell cultures, the proportion of neurons of specific layers differed from that in normal brains — additional biological evidence for the disorder. The neurons grown from the Timothy syndrome cells were less characteristic of lower-level neurons, which include neurons that bridge the left and right halves of the brain via the bundle of fibers known as the corpus callosum. This reinforces the view that autism results from defects in brain connectivity. 

Pasca and Dolmetsch had an "aha" moment when they realized the neurons grown from Timothy syndrome cells were making too much of the enzyme most critical for producing dopamine and norepinephrine, which play an important role in sensory processing and social behavior. The realization may offer important clues about what causes the problems seen in autism. 

To determine whether the enzyme upsurge was reversible, the scientists treated the neurons with a chemical that blocks the defective calcium channels, called roscovitine. They saw a nearly 70 percent reduction in the proportion of cells producing the enzyme, confirming the defective calcium channel was the culprit in producing too much dopamine and norepinephrine. Such reversibility suggests that certain cellular abnormalities in autism may be treatable. 

Dolmetsch warned, however, that roscovitine is not currently approved for use in humans and has never been tested in children. While it is currently in clinical trials for lung cancer, it reportedly causes nausea and other side effects. "The reported side effects are probably due to the fact that, in addition to targeting the channel that is mutated in autism, roscovitine also inhibits kinases that are required for cell proliferation," he said. "We think that roscovitine is a good starting point, but probably has to be optimized before it would be useful for autism." 

In the meantime, the study represents a major achievement with its success in developing a technique to recreate how the neurons of individuals with Timothy syndrome develop in a lab setting. It's the first time it's been possible to study the disorder in human cells rather than mouse cells, so it represents a better clinical model, Dolmetsch said. 

"These results could lead to a very powerful research tool," he said. "It's human psychiatric disease in a petri dish." 

Source: Stanford University Medical Center [November 27, 2011]

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Tuning out: How brains benefit from meditation

Experienced meditators seem to be able switch off areas of the brain associated with daydreaming as well as psychiatric disorders such as autism and schizophrenia, according to a new brain imaging study by Yale researchers. 

Experienced meditators seem to switch off areas of the brain associated with wandering thoughts, anxiety and some psychiatric disorders such as schizophrenia. Researchers used fMRI scans to determine how the brains of meditators differed from subjects who were not meditating. The areas shaded in blue highlight areas of decreased activity in the brains of meditators [Credit: Yale University]

Meditation's ability to help people stay focused on the moment has been associated with increased happiness levels, said Judson A. Brewer, assistant professor of psychiatry and lead author of the study published the week of Nov. 21 in the Proceedings of the National Academy of Sciences. Understanding how meditation works will aid investigation into a host of diseases, he said. 

"Meditation has been shown to help in variety of health problems, such as helping people quit smoking, cope with cancer, and even prevent psoriasis," Brewer said. 

The Yale team conducted functional magnetic resonance imaging scans on both experienced and novice meditators as they practiced three different meditation techniques. 

They found that experienced meditators had decreased activity in areas of the brain called the default mode network, which has been implicated in lapses of attention and disorders such as anxiety, attention deficit and hyperactivity disorder, and even the buildup of beta amyloid plaques in Alzheimer's disease. The decrease in activity in this network, consisting of the medial prefrontal and posterior cingulate cortex, was seen in experienced meditators regardless of the type of meditation they were doing. 

The scans also showed that when the default mode network was active, brain regions associated with self-monitoring and cognitive control were co-activated in experienced meditators but not novices. This may indicate that meditators are constantly monitoring and suppressing the emergence of "me" thoughts, or mind-wandering. In pathological forms, these states are associated with diseases such as autism and schizophrenia. 

The meditators did this both during meditation, and also when just resting — not being told to do anything in particular. This may indicate that meditators have developed a "new" default mode in which there is more present-centered awareness, and less "self"-centered, say the researchers. 

"Meditation's ability to help people stay in the moment has been part of philosophical and contemplative practices for thousands of years," Brewer said. "Conversely, the hallmarks of many forms of mental illness is a preoccupation with one's own thoughts, a condition meditation seems to affect. This gives us some nice cues as to the neural mechanisms of how it might be working clinically."  

Source: Yale University [November 21, 2011]

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