Invertebrates: A Few Exons Short of a Full Deck

Regulation of Vertebrate Nervous System Alternative Splicing and Development by an SR-Related Protein
Calarco, et. al. Cell 138: 898-910 (2009).

Most eukaryotic genes are comprised of functional modules, or exons, separated by intervening segments of DNA known as introns. After a gene is transcribed, introns are removed (or "spliced") out of the pre-mRNA by a large RNA-protein complex called the spliceosome, which recognizes characteristic nucleotide sequences at exon-intron boundaries. Other proteins can influence splicing by either promoting or repressing spliceosome formation at potential splice sites. This can lead to the inclusion of different combinations of exons in the mature mRNA, a phenomenon known as alternative splicing. The result of alternative splicing is that a single gene can encode multiple protein isoforms with different amino acid sequences. The fact that at least 95% of human genes are alternatively spliced is cited as one explanation for why humans have fewer genes than some less complex organisms.

An additional layer of complexity is added when expression of a factor that regulates splice site selection is restricted to a certain tissue, ensuring that a particular protein isoform is similarly tissue-specific. Several such splicing factors have already been identified, particularly in the mammalian nervous system, but in the September 4 issue of Cell, Calarco, et. al. identify a neural-specific splicing factor found only in vertebrates, suggesting that it may have played a role in the evolution of the vertebrate nervous system. One major way vertebrate nervous systems differ from their invertebrate counterparts is that they have a central component (i.e., a brain and a spinal cord). Another is that their brains contain several specialized regions dedicated to particular functions. Surprisingly, the brains of all vertebrates have the exact same number of brain divisions (except jawless fish which lack a true cerebellum). Therefore, it is thought that the general structure of what we call a "brain " must have evolved at the same time as the first vertebrate, or shortly thereafter.

Many proteins involved in the regulation of alternative splicing have a characteristic arginine/serine (RS or SR) repeat domain, so in order to find potentially novel splicing factors, the authors used BLAST to identify all mouse protein-encoding genes with RS domains. They then analyzed the expression pattern of the 112 genes they found across a diverse panel of cell and tissue types, identifying a subset of RS domain genes whose expression was restricted to the adult nervous system. The authors chose to focus on one gene whose distinguishing feature is that it is highly conserved only in vertebrates. The authors demonstrate via RT-PCR, immunofluorescence, and Western blot that this gene is highly expressed only in neurons of the brain and sensory organs and encodes a 100 kilodalton protein, and consequently name it nSR100 (or neural-specific SR related protein of 100 kD).

The authors then utilize a custom microarray to determine which alternative splicing events are most affected following nSR100 knockdown. This lab previously developed this strategy using probes for both exon bodies and exon-intron junctions to detect the frequency of common alternative splicing events in different cell and tissue types. Using this technique, they show that loss of nSR100 in a neuronal cell line results in increased exon skipping, suggesting nSR100 normally acts to promote alternative exon inclusion in neurons. The authors verify by exon- specific RT-PCR that nSR100 knockdown does increase exon exclusion in the genes identified on the microarray, as well as show that nSR100 can bind mRNA containing target exons prior to splicing. Excitingly, they also show that nSR100 has a conserved function in neural development, as it is required for differentiation of neural stem cells in mice as well as nervous system development in zebrafish embryos.

The exact mechanism of how nSR100 is promoting alternative exon inclusion remains unclear, though the authors would argue its through antagonism of a known repressor of exon inclusion. Their data also suggests that nSR100 is necessary, but not sufficient, for exon inclusion, indicating a role for other splicing factors in nSR100-mediated regulation of alternative splicing. Furthermore, it remains to be seen what the functional consequences of the nSR100-dependent alternative splicing events are, though the authors point out that protein-protein interaction domains are encoded by some of the exons and that the literature suggests that the affected proteins may be part of an interaction network regulating cytoskeleton remodeling during neuronal differentiation. If that's true, it may be the mind-boggling number of combinations of alternative splicing events that allow a vertebrate's mind to be boggled.

Other References:
Northcutt, R.G. Understanding Vertebrate Brain Evolution. Integrative and Comparative Biology 42(4):743-756 (2002).
Pan, Q. Revealing Global Regulatory Features of Mammalian Alternative Splicing Using a Quantitative Microarray Platform. Molecular Cell 16(6): 929-941 (2004).