Expanding on prior research performed at the University of Pennsylvania, Penn biologists have determined that faulty RNA, the blueprint that creates mutated, toxic proteins, contributes to a family of neurodegenerative disorders in humans.

Nancy Bonini, professor in the Department of Biology at Penn and an investigator of the Howard Hughes Medical Institute, and her team previously showed that the gene that codes for the ataxin-3 protein, responsible for the inherited neurodegenerative disorder Spinocerebellar ataxia type 3, or SCA3, can cause the disease in the model organism Drosophila. SCA3 is one of a class of human diseases known as polyglutamine repeat diseases, which includes Huntington’s disease. Previous studies had suggested that the disease is caused largely by the toxic polyglutamine protein encoded by the gene.

The current study, which appears in the journal Nature, demonstrates that faulty RNA, the blueprint for the toxic polyglutamine protein, also assists in the onset and progression of disease in fruit fly models.

“The challenge for many researchers is coupling the power of a simple genetic model, in this case the fruit fly, to the enormous problem of human neurodegenerative disease,” Bonini said. “By recreating in the fly various human diseases, we have found that, while the mutated protein is a toxic entity, toxicity is also going on at the RNA level to contribute to the disease.”

To identify potential contributors to ataxin-3 pathogenesis, Bonini and her team performed a genetic screen with the fruit fly model of ataxin-3 to find genes that could change the toxicity. The study produced one new gene that dramatically enhanced neurodegeneration. Molecular analysis showed that the gene affected was muscleblind, a gene previously implicated as a modifier of toxicity in a different class of human disease due to a toxic RNA. These results suggested the possibility that RNA toxicity may also occur in the polyglutamine disease situation.

The findings indicated that an RNA containing a long CAG repeat, which encodes the polyglutamine stretch in the toxic polyglutamine protein, may contribute to neurodegeneration beyond being the blueprint for that protein. This raised the possibility that expression of the RNA alone may be damaging.

Long CAG repeat sequences can bind together to form hairpins, dangerous molecular shapes. The researchers therefore tested the role of the RNA by altering the CAG repeat sequence to be an interrupted CAACAG repeat that could no longer form a hairpin. Such an RNA strand, however, would still be a blueprint for an identical protein. The researchers found that this altered gene caused dramatically reduced neurodegeneration, indicating that altering the RNA structure mitigated toxicity. To further implicate the RNA in the disease progression, the researchers then expressed just a toxic RNA alone, one that was unable to code for a protein at all. This also caused neuronal degeneration. These findings revealed a toxic role for the RNA in polyglutamine disease, highlighting common components between different types of human triplet repeat expansion diseases. Such diseases include not only the polyglutamine diseases but also diseases like myotonic dystrophy and fragile X.

The family of diseases called polyglutamine repeat disorders arise when the genetic code of a CAG repeat for the amino acid glutamine stutters like a broken record within the gene, becoming very long. This leads to an RNA — the blueprint for the protein — with a similar long run of CAG. During protein synthesis, the long run of CAG repeats are translated into a long uninterrupted run of glutamine residues, forming what is known as a polyglutamine tract. The expanded polyglutamine tract causes the errant protein to fold improperly, leading to a glut of misfolded protein collecting in cells of the nervous system, much like what occurs in Alzheimer’s and Parkinson’s diseases.

Polyglutamine disorders are genetically inherited ataxias, neurodegenerative disorders marked by a gradual decay of muscle coordination, typically appearing in adulthood. They are progressive diseases, with a correlation between the number of CAG repeats within the gene, the severity of disease and age at onset.

In addition to Bonini, researchers whose work contributed to this study are Ling-Bo Li, formerly in the Department of Biology at Penn and now with the Department of Biochemistry at the University of Utah, and Zhenming Yu and Xiuyin Teng of the Department of Biology at Penn and the Howard Hughes Medical Institute.

Funding for this study was provided by the National Institute of Neurological Disorders and Stroke.

Illinois postdoctoral researcher Feng-Jie Sun (left) and crop sciences professor Gustavo Caetano-Anollés began with the idea that understanding the structural properties of tRNA would shed light on how organisms and viruses evolved. Photo by L. Brian Stauffer, U. of I. News Bureau.

Transfer RNA (also called tRNA) is an ancient molecule, central to every task a cell performs and thus essential to all life. A new study from the University of Illinois indicates that it is also a great historian, preserving some of the earliest and most profound events of the evolutionary past in its structure.

The study, co-written by Gustavo Caetano-Anollés, a professor of crop sciences, and postdoctoral researcher Feng-Jie Sun, appears March 7 in PLoS Computational Biology. Caetano-Anollés is an affiliate of the U. of I. Institute for Genomic Biology.

Of the thousands of RNAs so far identified, transfer RNA (tRNA) is the most direct intermediary between genes and proteins. Like many other RNAs (ribonucleic acids), tRNA aids in translating genes into the chains of amino acids that make up proteins. With the help of a highly targeted enzyme, each tRNA molecule recognizes and latches onto a specific amino acid, which it carries into the protein-building machinery. In order to successfully add its amino acid to the end of a growing protein, tRNA must also accurately read a coded segment of messenger RNA, which gives instructions for the exact sequence of amino acids in the protein.

The fact that tRNA is so central to the task of building proteins probably means that it has been around for a long time, Caetano-Anollés said. His inquiry began with a hunch that understanding the structural properties of tRNA would shed light on how organisms and viruses evolved.

“Perhaps in evolution there are things that are so fundamental that they are kept, held onto, for millions or even billions of years,” Caetano-Anollés said. “Those are the fossils, the molecular fossils, that tell us about the past. Therefore, studying these molecules can address fundamental questions in biology and evolution.”

All tRNAs assemble themselves into a shape that, if flattened, resembles a cloverleaf. Patterns in these structures give clues to early evolutionary history. The red areas of the molecule pictured above are the most ancient. Image courtesy of Gustavo Caetano-Anollés.

All tRNAs assemble themselves into a shape that, if flattened, resembles a cloverleaf. The team began by looking for patterns in this cloverleaf structure, using detailed data from hundreds of molecules representing viruses and each of the three superkingdoms of life: archaea, bacteria and eukarya.

The researchers converted all distinguishing features of the individual tRNA cloverleaf structures into coded characters, a process that allowed a computerized search for the most “parsimonious” (that is, the simplest, most probable) tRNA family tree. They conducted the same analysis on the tRNAs of each of the superkingdoms, to see how far these groupings diverged from the overall tree. This comparison allowed them to determine the order in which viruses and each of the superkingdoms diverged.

The new analysis supports an earlier study that suggested that the archaea were the first to arise as an evolutionarily distinguishable group. Archaea are microbes that can survive in boiling acid, near sulfurous ocean vents or in other extreme environments. The earlier study, also led by Caetano-Anollés, analyzed the vast catalog of protein folds – those precisely configured regions in proteins that give them their functionality – as a guidebook to evolutionary history.

“The transfer RNA data matches our earlier data,” Caetano-Anollés said. “This is important because two lines of independent evidence are supporting each other.”

The new analysis also indicates that viruses emerged not long after the archaea, with the superkingdoms eukarya and bacteria following much later – and in that order. This finding may influence the ongoing debate over whether viruses existed prior to, or after, the emergence of living cells, Caetano-Anollés said.

“This supports the idea that viruses arose from the cellular domain,” he said.