MIT scientists report that because alternative RNA splicing often alters the outputs of signaling networks in different tissues, this may be largely responsible for the differences observed among various species.

After analyzing vast amounts of genetic data, the researchers found that the same genes are expressed in the same tissue types, such as liver or heart, across mammalian species. However, alternative splicing patterns, which determine the segments of those genes included or excluded, vary from species to species.

“The core things that make a heart a heart are mostly determined by a heart-specific gene expression signature. But the core things that make a mouse a mouse may disproportionately derive from splicing patterns that differ from those of rats or other mammals,” says Chris Burge, Ph.D., an MIT professor of biology and biological engineering, and senior author of a paper on the findings in the December 20 online edition of Science.

Alternative RNA splicing controls the composition of proteins encoded by a gene. In mammals, genes consist of many short segments known as exons and introns. After the DNA is copied into an RNA transcript, all introns and frequently some exons are excised before the messenger RNA (mRNA) leaves the nucleus, carrying instructions to make a specific protein. This process allows cells to create a much wider variety of proteins than would be possible if each gene encoded only one protein.

In 2008, Dr. Burge and colleagues analyzed mRNA from 10 different human tissues, publishing their results in Nature, and found that nearly every gene is alternatively spliced. Furthermore, most alternative splicing was found to differ among tissues. In the new study, the researchers compared tissues from several different mammalian species (the rhesus monkey, rat, mouse, and cow) as well as one species of bird, the chicken. For each species, the researchers analyzed nine types of tissue (brain, colon, heart, kidney, liver, lung, muscle, spleen, and testes) from three individuals, sequencing more than a trillion bases of mRNA.

Using new high-speed sequencing technology, the researchers analyzed both gene expression and alternative splicing patterns in each tissue sample. They found that gene-expression patterns were extremely similar across tissues, no matter what species the tissue came from. That is, the genes active in kidney tissue from rats were nearly identical to those turned on in cows’ kidney tissue.

“That was not a big surprise,” points out Dr. Burge. “It’s consistent with the idea that the gene expression pattern actually determines the identity of the tissue. You need to express certain structural and motor proteins if you’re a muscle cell, and if you’re a neuron you have to express certain synaptic proteins.”

The results from the alternative splicing pattern comparison were very different. Instead of clustering by tissue, the patterns clustered mostly by species. “Different tissues from the cow look more like the other cow tissues, in terms of splicing, than they do like the corresponding tissue in mouse or rat or rhesus,” according to Dr. Burge.

Because splicing patterns are more specific to each species, it appears that splicing may contribute preferentially to differences between those species, he continues. “Splicing seems to be more malleable over shorter evolutionary timescales, and may contribute to making species different from one another and helping them adapt in various ways.”

The researchers also found that a major function of alternative splicing is the addition and deletion of short protein segments that contain one or more phosphorylation sites. Phosphorylation is a common way for cells to activate or deactivate proteins. When a variant form of a protein lacks a key phosphorylation site, it may lose the function of the original form. Phosphorylation can also direct proteins to different locations within the cell, which may alter their function.

Changes in splicing patterns also help to modify the signaling networks that regulate most cellular activity. These networks are often controlled by phosphorylation of proteins involved in the network, many of which can be alternatively spliced. “You can think about it as rewiring signaling networks so they control different outputs. Splicing can add a new output or delete it in a tissue-specific way,” explains Dr. Burge.

The researchers also identified several thousand new alternative exons in each species, and are now studying how these exons evolved and exploring their potential functions.

The specific importance of signaling and alternative splicing, which led to differences in the brain among primates, was demonstrated by Lin et al at the University of Iowa (online, May 11, 2010, Human Molecular Genetics). After profiling the brain transcriptomes of humans, chimpanzees, and rhesus macaques, the scientists concluded that evolution of cis-regulatory signals is a major contributor to the emergence of human-specific splicing patterns. “Together, our data reveal widespread human-specific changes of alternative splicing in the brain and suggest an important role of splicing in the evolution of neuronal gene regulation and functions,” wrote the researchers.

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