A ripple effect: skipping a single exon in PTBP1 leads to changes in splicing and neural differentiation


What makes a human, human? Or a chicken, chicken?

The preeminent belief has been that the difference between species lies in their DNA—the number of genes an organism has, the function of those genes and when and where those genes are expressed. As it turns out, the answer is not quite so simple.

“There’s very high conservation of the total number of protein coding genes across different vertebrate species,” says Serge Gueroussov, a PhD student in Dr. Benjamin Blencowe’s lab at the University of Toronto. “When [researchers] compared gene expression across different organs in different species, there was also a lot of conservation. It suggests that organisms don’t differ so much in the genes they have and the extent to which they express [those genes].”

In other words, while we may look drastically different from a frog or a chicken, our repertoire of genes and when and where we express those genes are actually pretty similar. So where is the variation coming from?

The answer may lie, in part, in a process called alternative splicing.

In most organisms, genes are broken up into fragments called exons. Interspersed between exons are regions of DNA known as introns. In order for the gene to be properly transcribed into a message, the introns must be removed so that all the exons are arranged together side-by-side. Think of it as removing all the extra spaces between your words to form a sentence that’s clear and easy to read. This process is called splicing. It happens in pretty much all living organisms except bacteria and viruses.

Sometimes an exon is removed along with the intron causing the remaining exons to be joined together in a new way. Because of this, one gene can have the potential to create many different messages because different exons can be removed from the message. These messages are then decoded to make proteins that differ from each other in just the slightest way. Imagine taking your sentence from earlier and creating five new sentences by randomly deleting a single word from the original sentence. Some words you can delete with little consequence while other deletions can dramatically change the meaning of the original sentence. The same is true for these “same, same but different” proteins called isoforms.

A 2012 study from Blencowe’s group showed that among the vertebrate animals (those with a spine), alternative splicing happens more frequently as you move closer to the primates. “Splicing has diverged much more than gene expression,” says Gueroussov, who worked on the 2012 study. This finding led them to hypothesize that splicing and the unique protein isoforms that have evolved as a result of alternative splicing are important contributors to the development of observable differences between species.

The researchers tested their hypothesis by studying a protein called PTBP1. All vertebrates have PTBP1—its DNA sequence is well conserved throughout. Within the cell, PTBP1 primarily functions as a regulator of splicing and is itself alternatively spliced. In mammals, one exon is cut out of the message leading to a slightly shorter version of PTBP1.

To understand the effects of excluding that particular exon, Gueroussov forced mouse cells to express the full-length PTBP1 protein. He found that in these cells, full-length PTBP1 was a much stronger regulator of splicing than the slightly shorter PTBP1 found in mammals.

One of the main roles for PTBP1 is regulating the splicing of genes important for neural differentiation, the process whereby stem cells develop into the cells that will later become our brain and nervous system. Full-length PTBP1 represses the network of genes that are required for neural differentiation. Because it’s a weaker regulator, the shorter mammalian-specific PTBP1 could make it easier to activate this genetic network and initiate the differentiation process. In support of this idea, Gueroussov found that expression of full-length PTBP1 caused mouse stem cells to alter their gene expression such that neural differentiation was significantly delayed.

Talk about a ripple effect: getting rid of that one exon in mammalian PTBP1 was enough to cause changes in gene expression that ultimately affected the transition of stem cells to neuronal cells. “I was surprised that manipulating one exon through this one [exon] skipping event could have pretty dramatic effects on neural differentiation,” says Gueroussov.

What’s more, when he expressed the mammalian form of PTBP1 in chicken cells, which normally express only the full length PTBP1, the slightly shorter PTBP1 caused the chicken cells to activate a similar set of genes as PTBP1 did in its native mammalian cells. “With the chicken cells, we showed that even deletion of one exon [from full length chicken PTBP1] can have dramatic effects on [alternative splicing of other genes]—I think that’s quite a cool result,” he says.

Why would mammals have evolved to keep the shorter form of PTBP1 even though it’s a weaker regulator? Gueroussov speculates that having both a strong and a weak version of PTBP1 may allow greater precision in fine-tuning the timing of the neural differentiation process. He is now looking at other splicing regulators that have species-specific isoforms to see if those isoforms, like the mammalian PTBP1, have also evolved new functions in development.

Reference: Gueroussov S, Gonatopoulos-Pournatzis T, Irimia M, Raj B, Lin ZY, Gingras AC, & Blencowe BJ (2015). RNA SPLICING. An alternative splicing event amplifies evolutionary differences between vertebrates. Science (New York, N.Y.), 349 (6250), 868-73 PMID: 26293963


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