In 2015, Bañez-Coronel and colleagues reported that RAN proteins are found in human HD autopsy brains near proteins associated with cell death and inflammation. These proteins are toxic in cells and are present in brain regions affected in HD. This is important for SCAs because, like HD, many SCAs are also caused by CAG repeat expansions and could have similar pathology.

It is crucial to investigate the role of mutant RNA and RAN proteins produced by CAG repeat expansions. That way, we can learn if and how these molecules function in disease. Das and colleagues began to tease out the contributions of repeat RNA and RAN proteins by looking at where both were located in the cell. This was done by expressing the repeat RNA and RAN proteins in cells.

To begin, the authors observed that the DNA sequences surrounding the CAG repeat expansion are vital for RAN translation. This means that the sequences that come before and after the repeat influence whether RAN proteins are made or not. They further found that mutant RNA formed clusters in both the nucleus and the cytoplasm. Not only that, but RAN proteins caused RNA to cluster just outside the nucleus of the cell. After discovering these RNA-RAN protein clusters, or aggregates, the Das and colleagues group answered two main questions:

• Do RNA-RAN aggregates trap proteins involved in RNA regulation?
• Do RNA-RAN aggregates interfere with transport between the nucleus and the rest of the cell?

The researchers found that proteins associated with protein aggregation and neurodegenerative disease cluster with RNA-RAN aggregates. These proteins—p62, TDP-43, and FUS—aggregate or are not in the right place in multiple neurodegenerative diseases. This pathology is correlated with disease, and seeing their interaction with RNA-RAN aggregates shows that they might be harmful to the cell and could cause the cell to die.

Another method of disrupting cell function is by interfering with transport. The nucleus in a cell is constantly sending and receiving cargo. The doors used to shuttle the cargo are called nuclear pores. Nuclear pores surround the nucleus and are essential for proper cell functioning. Since RNA-RAN aggregates gather around the nucleus, it is imperative to consider the impact on nuclear-cytoplasmic transport.

To investigate this question, the authors looked at a protein called RanGTP1, which is involved in nuclear transport. They found that this protein was not in the place it was supposed to be around the nucleus. The authors further observed the flow of molecules in and out of the nucleus and saw that a lot of molecules that should be entering the nucleus were not able to enter. Interruption of transport in and out of the nucleus is extremely harmful to the cell and could lead to cell stress and increased levels of cell death.

RNA-RAN protein aggregates are harmful to cells and trap RNA regulator proteins as well as disturb transport of nuclear-cytoplasmic contents. The next question the authors asked was, what molecule is driving toxicity in these cells? Is it mostly the repeated RNA, or is it mostly the RAN proteins? In pursuit of this answer, the Das and colleagues designed molecules that bind to the mutant RNA and prevent RAN translation. This caused a significant decrease in RAN protein levels and cytoplasmic RNA clusters. Preventing RAN translation allowed TDP-43 to be localized to its proper place in the cell and reduced cell death. These results show that RAN proteins play an important role in cell toxicity and that preventing the translation of RAN proteins helps the cells function better.

RAN translation in the field of spinocerebellar ataxia is still relatively new. Historically in CAG repeat expansion diseases, the focus has been on the impact of polyglutamine-expanded proteins and how their functions change with the repeat expansion. Researchers are actively working to uncover the role of RAN proteins in CAG-expanded SCAs. These toxic proteins provide ideal therapeutic targets that, when cleared, could mitigate disease in many SCAs someday.