Non-coding RNA has role in inherited neurological disorder -- and maybe other brain diseases too

Non-coding RNA has role in inherited neurological disorder -- and maybe other brain diseases too

  1:26:00 AM    Science Lover

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A team of scientists, led by researchers at the University of California, San Diego School of Medicine, have uncovered a novel mechanism regulating gene expression and transcription linked to Spinocerebellar ataxia 7, an inherited neurological disorder. The discovery promises to have broad ramifications, suggesting that abundant non-coding transcripts of ribonucleic acid (RNA) may be key players in neurological development and function, and could be powerful targets for future clinical therapies.

Researchers have discovered that expression
of the ataxin‑7 gene - the cause of the
neurological disorder spinocerebellar
ataxia type 7 - has two regulators: a highly
conserved, multi‑tasking protein called
CTCF and, surprisingly, an adjacent promoter
containing non‑coding RNA.
Credit: Illustration courtesy of Christina
Takamatsu‑Butler, UC San Diego.

The research, headed by Albert La Spada, MD, PhD, chief of the division of genetics in the UCSD department of pediatrics, and professor of cellular and molecular medicine, neurosciences and biological sciences, is published in the June 22 issue of the journal Neuron.

"Our paper highlights a number of important emerging themes in our understanding of gene regulation in the brain," said La Spada, who is also associate director of the UCSD Institute for Genomic Medicine.

"With the advent of new technologies, science has learned that the vast majority of our transcripts are non-coding," said La Spada. "The challenge going forward is to determine what they do do, and if they have specific functions. It now seems increasingly likely that a multitude of these non-coding RNAs help finely tune transcription regulation in the brain, and perturbation of their work is linked to disease. If we can figure out exactly how, we should be able to gain new insights into how the brain is so precisely regulated – knowledge that may help us better understand how the brain works."



Spinocerebellar ataxia 7 is one of several types of spinocerebellar ataxia (SCA), genetic degenerative disorders characterized by atrophy in the cerebellum of the brain, progressive loss of physical coordination – and in the case of type 7 – retinal degeneration that can result in blindness. There is currently no known cure.

Many SCAs are classified as polyglutamine diseases, caused when a protein associated with the disease contains too many repeats of the amino acid glutamine. Polyglutamine diseases are also known as "CAG Triplet Repeat Disorders" because CAG is the sequence of nucleic acids that codes for glutamine.

La Spada and colleagues have long studied SCA. In 2001, they were the first to demonstrate that SCA7 retinal degeneration was the result of transcription dysregulation of ataxin-7, the protein associated with SCA7. Following up, they decided to learn how the gene that expresses ataxin-7 is itself regulated.

The researchers found not one, but two, regulators. The first is called CTCF, a highly conserved protein that regulates a variety of transcriptional processes, most notable establishing insulator domains and controlling genomic imprinting. But they also discovered an adjacent, alternative promoter dubbed intron 2 promoter (P2A) and a transcribed antisense, non-coding RNA, which they labeled SpinoCerebellarAtaxia-AntisenseNoncodingTranscript1 or SCAANT1.

Antisense RNA is single-stranded ribonucleic acid whose primary function appears to be as an inhibitor or suppressor of a gene, though sometimes it can promote gene expression instead. Most antisense RNAs are non-coding, meaning that their sequences do not provide information for making proteins. Even though non-coding RNAs do not provide instructions for the production of vital proteins, they comprise the bulk of the human genome. A major challenge for biomedical research in the 21st century is to figure what they do, and how they do it.

In their Neuron paper, La Spada and colleagues highlight one function, at least for SCAANT1. When they investigated how CTCF regulated ataxin-7 gene expression in transgenic mice, they discovered that CTCF promotes the production of SCAANT1 which in turn represses the newly discovered ataxin-7 sense promoter P2A. In mice lacking SCAANT1, sense promoter P2A is de-repressed, allowing a mutant ataxin-7 gene to be expressed, resulting in mice with a version of SCA7. The scientists found a similar lack of antisense SCAANT1 in the fibroblasts and white blood cells taken from human patients with SCA7, implicating deregulation of this pathway in the disease process.

As many inherited neurological disorders are now known to exhibit such overlapping "bidirectional" transcription, the findings in SCA7 could shed light on similar abnormalities with non-coding RNA function in a number of brain diseases.

Provided by University of California - San Diego

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Biological Circuits for Synthetic Biology

Biological Circuits for Synthetic Biology

  1:14:00 AM    Science Lover

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"If you don't like the news, go out and make some of your own," said Wes "Scoop" Nisker. Taking a page from the book of San Francisco radio legend Scoop Nisker, biologists who find themselves dissatisfied with the microbes nature has provided are going out and making some of their own. Members of the fast-growing "synthetic biology" research community are designing and constructing novel organisms and biologically-inspired systems -- or redesigning existing organisms and systems -- to solve problems that natural systems cannot. The range of potential applications for synthetic biological systems runs broad and deep, and includes such profoundly important ventures as the microbial-based production of advanced biofuels and inexpensive versions of critical therapeutic drugs.

Berkeley Lab researchers are using RNA molecules 
to engineer genetic networks – analogous to 
microcircuits - into E. coli. (Credit: Image courtesy of 
DOE/Lawrence Berkeley National Laboratory)

Synthetic biology, however, is still a relatively new scientific field plagued with the trial and error inefficiencies that hamper most technologies in their early stages of development. To help address these problems, synthetic biologists aim to create biological circuits that can be used for the safer and more efficient construction of increasingly complex functions in microorganisms. A central component of such circuits is RNA, the multipurpose workhorse molecule of biology.

"A widespread natural ability to sense small molecules and regulate genes has made the RNA molecule an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering," says Adam Arkin, a computational biologist with the U.S. Department of Energy (DOE)'s Lawrence Berkeley National Laboratory (Berkeley Lab), where he serves as director of the Physical Biosciences Division. Arkin is also a professor at the University of California (UC) Berkeley where he directs the Synthetic Biology Institute, a partnership between UC Berkeley and Berkeley Lab.

In his multiple capacities, Arkin is leading a major effort to use RNA molecules for the engineering of programmable genetic networks. In recent years, scientists have learned that the behavior of cells is often governed by multiple different genes working together in networked teams that are regulated through RNA-based mechanisms. Synthetic biologists have been using RNA regulatory mechanisms to program genetic networks in cells to achieve specific results. However, to date these programming efforts have required proteins to propagate RNA regulatory signals. This can pose problems because one of the primary goals of synthetic biology is to create families of standard genetic parts that can be combined to create biological circuits with behaviors that are to some extent predictable. Proteins can be difficult to design and predict. They also add a layer of complexity to biological circuits that can delay and slow the dynamics of the circuit's responses.

"We're now able to eliminate the protein requirement and directly propagate regulatory signals as RNA molecules," Arkin says.

Working with their own variations of RNA transcription attenuators -- nucleotide sequences that under a specific set of conditions will stop the RNA transcription process -- Arkin and his colleagues engineered a system in which these independent attenuators can be configured to sense RNA input and synthesize RNA output signals. These variant RNA attenuators can also be configured to regulate multiple genes in the same cell and -- through the controlled expression of these genes -- perform logic operations.

"We have demonstrated the ability to construct with minimal changes orthogonal variants of natural RNA transcription attenuators that function more or less homogeneously in a single regulatory system, and we have shown that the composition of this system is predictable," Arkin says. "This is the first time that the three regulatory features of our system, which are all properties featured in a semiconductor transistor, have been captured in a single biological molecule."

A paper describing this breakthrough appears in the Proceedings of the National Academy of Science (PNAS).

The success of Arkin and his colleagues was based on their making use of an element in the bacterial plasmid (Staphylococcus aureus) known as pT181. The element in pT181 was an antisense RNA-mediated transcription attenuation mechanism that controls the plasmid's copy number. Plasmids are molecules of DNA that serve as a standard tool of synthetic biology for, among other applications, encoding RNA molecules. Antisense RNA consists of non-coding nucleotide sequences that are used to regulate genetic elements and activities, including transcription. Since the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism works through RNA-to-RNA interactions, Arkin and his colleagues could use it to create attenuator variants that would independently regulate the transcription activity of multiple targets in the same cell -- in this case, in Escherichia coli, one of the most popular bacteria for synthetic biology applications.

"It is very advantageous to have independent regulatory units that control processes such as transcription because the assembly of these units into genetic networks follows a simple rule of composition," Arkin says.

While acknowledging the excellent work done on other RNA-based regulatory mechanisms that can each perform some portion of the control functions required for a genetic network, Arkin believes that the attenuator variants he and his colleagues engineered provide the simplest route to achieving all of the required control functions within a single regulatory mechanism.

"Furthermore," he says, "these previous efforts were fundamentally dependent on molecular interactions through space between two or more regulatory subunits to create a network. Our approach, which relies on the processive transcription process, is more reliable."

Arkin and his colleagues say their results provide synthetic biologists with a versatile new set of RNA-based transcriptional regulators that could change how future genetic networks are designed and constructed. Their engineering strategy for constructing orthogonal variants from natural RNA system should also be applicable to other gene regulatory mechanisms, and should add to the growing synthetic biology repertoire.

"Although RNA has less overall functionality than proteins, its nucleic acid-based polymer physics make mechanisms based on RNA simpler and easier to engineer and evolve," Arkin says. "With our RNA regulatory system and other work in progress, we're on our way to developing the first complete and scalable biological design system. Ultimately, our goal is to create a tool revolution in synthetic biology similar to the revolution that led to the success of major integrated circuit design and deployment."

Much of this research was supported by was supported by the Synthetic Biology Engineering Research Center (SynBERC) under a grant from the National Science Foundation.

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