How RNA regulates sex-linked disorders

Boys have an X and a Y chromosome inside each cell. Girls have two X's. That small difference explains why boys are more at risk for disorders, from autism to hemophilia, linked to gene defects on their one X chromosome. We may now know how parts of that X can be silenced — to better treat X-linked diseases.

Most girls who carry the same defect on one of their X chromosomes as a boy with the same X chromosome, won't get sick. This is because their other X chromosome takes over. It becomes the "back-up" X whose genes function, while the other X is "silenced". A new gene discovery helps scientists explain how this happens.

In a new paper in Nature Communications, scientists from the University of Michigan Medical School show that in female cells — not male cells — tiny amounts of RNA are made that silence the defective X chromosome. They call it 'Xist Activating RNA' — or XistAR.

Back in high school biology, you probably learned that RNAs make proteins in cells. But XistAR is special. It belongs to a class of RNAs called long noncoding RNAs — or lncRNAs. These RNAs don't make proteins, they control how genetic code is read.

U-M geneticist Sundeep Kalantry PhD, and his team knew the gene called Xist causes cells to make a version of lncRNA that coats an X and keeps it dormant or "silenced".

But how was Xist lncRNA itself made?

Genes can be 'read' in both a forward and backward direction which increases their functionality. These molecules are held together by electro-magnetic fields, and although they have the same molecular formula and sometimes appear in the same sequence, they can have a different arrangement of atoms and so have different properties.

The Kalantry team found that the Xist gene on a silent X chromosome is read in both a forward and backward direction simultaneously.

XistAR read in a backward direction generates movement as does reading it in a forward direction. However, perpetual forward and back reading of XistAR keeps it sealed away — effectively turning off the X chromosome.

To prove their discovery, the team developed a new test to find backward read RNA strands, which cells make in much smaller amounts than forward-read RNA strands. The test can now search through the genome for more examples of rare lncRNAs — made in the backward or 'antisense' direction.

Experimenting in mice, scientists could follow what happens when they stop cells from making XistAR. Without XistAR, cells couldn't silence one of the two female X chromosomes which resulted in too many X-chromosome gene products.

If this discovery translates to humans — and XistAR seems to be present in humans — Kalantry believes it may provide a convenient handle to manipulate otherwise defective genes on X chromosomes. For example, it might be possible to boost proteins inaccessible in defective genes carried in some male X chromosomes. Kalantry's team is now looking for factors to control XistAR.

"This work sheds light into how lncRNAs function, how genes and even an entire chromosome can be quieted. XistAR provides a molecular target to control gene expression — either how to 'wake the genes up' or reduce their activity.

"Exploring how the X chromosome becomes inactivated lets us know how to selectively make it activate. Turning on a healthy copy of an X chromosome gene may be a way to minimize disease risks associated with the X chromosome."

Sundeep Kalantry PhD, Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA

Meanwhile, Kalantry says, understanding how antisense lncRNAs work in an "epigenetic" way — as an outside force controlling what gene gets read or not — applies far beyond the X-chromosome.

Calico cats and autism

Kalantry, an assistant professor in the U-M Department of Human Genetics, keeps a portrait of a calico cat on his office wall to remind him of the power of the X-chromosome inactivation.

Calicos, nearly all female, have patches of fur in different colors growing from different regions of their skin. Each patch of fur depends on which X-chromosome has been turned off or on in their hair-producing cells — one X inherited from the mother or one X from the father. Inactivating the doppelganger X, also called Lyonization, happens at the earliest stages of embryo development. Once each cell in the tiny clump of female embryo cells has "decided" which X to silence, all cells forever inactivate copies of that same X.

But in humans, the effects of in-activating a "healthy" X chromosome more often than an "unhealthy" X — can really change a girl's life. For example, Rett syndrome is an autism-like disorder occuring only in girls. It occurs when a mutated gene on one of her two X chromosomes is 'turned on'. Autism is heavily linked to genetic problems on the X-chromosome. This explains why boys with only one X chromosome are four times more likely than girls to be on the autistic spectrum.

What Happens Next?

Strategies to block RNA strands in cells are already being tested in clinical trials. This paves the way for future research into treating autism or Rett syndrome by altering the activity of "unhealthy" X chromosomes.

Kalantry notes his team has developed ways to find short-lived lncRNAs such as XistAR and "tag" such strands to make them detectable.

Kalantry: "The control of genes by lncRNAs is often via epigenetic influences, now realized to occur in a wide variety of contexts from normal physiology to disease. On a fundamental level, lncRNAs reverse the central dogma of DNA begetting RNA, which then make proteins. Techniques we've developed facilitate the discovery of rare RNA species in a cell. Such RNAs, to date, have been missed by high-throughput gene sequencing although essential to cell function."

Abstract
The transcriptional imbalance due to the difference in the number of X chromosomes between male and female mammals is remedied through X-chromosome inactivation, the epigenetic transcriptional silencing of one of the two X chromosomes in females. The X-linked Xist long non-coding RNA functions as an X inactivation master regulator; Xist is selectively upregulated from the prospective inactive X chromosome and is required in cis for X inactivation. Here we discover an Xist antisense long non-coding RNA, XistAR (Xist Activating RNA), which is encoded within exon 1 of the mouse Xist gene and is transcribed only from the inactive X chromosome. Selective truncation of XistAR, while sparing the overlapping Xist RNA, leads to a deficiency in Xist RNA expression in cis during the initiation of X inactivation. Thus, the Xist gene carries within its coding sequence an antisense RNA that drives Xist expression.

The research was funded by the National Institutes of Health, including a New Innovator Award to Kalantry (OD008646). Three post-doctoral fellows share first authorship on the study: Mrinal Kumar Sarkar, Ph.D., Srimonta Gayen, Ph.D., and Surinder Kumar, Ph.D.

Reference: Nature Communications, DOI: 10.1038/ncomms9564, http://www.nature.com/ncomms/2015/151019/ncomms9564/full/ncomms9564.html

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Oct 23, 2015   Fetal Timeline   Maternal Timeline   News   News Archive   

In these [BLUE] mouse embryo cells, XistAR RNA strands show up as RED
— the Xist as GREEN. Scientists have developed this new method to detect
antisense lncRNA in order to identify, and someday affect production of, XistAR RNA.
Image Credit: University of Michigan