Microexons, great new drugable targets

Some very serious new players in cellular and tissue molecular biology have just been found. They are very juicy drugable targets, not that targeting them will be easy. If you don’t know what introns, exons and alternate splicing are, it’s time to learn. Go to https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ read and follow the links forward. It should be all you need to comprehend the following.

The work came out at the tail end of 2014 [ Cell vol. 159 pp. 1488 – 1489, 1511 -1523 ’14 ]. Microexons are defined as exons containing 50 nucleotides or less (the paper says 3 – 27 nucleotides). They have been overlooked, partially because their short length makes them computationally difficult to find. Also few bothered to look for them as they were thought to be unfavorable for splicing because they were too short to contain exonic splicing enhancers. They are so short that it was thought that the splicing machinery (which is huge) couldn’t physically assemble at both the 3′ and 5′ splice sites. So much for theory, they’re out there.

What is a cell and tissue differentially regulated alternative splicing event? It’s the way a given mRNA can be spliced together one way in tissue/cell #1 and another in tissue/cell #2 producing different proteins in each. Exons subject to tissue specific alternative splicing are significantly UNDERrepresented in well folded domains in proteins. Instead they are found in regions of protein disorder more frequently than one would expect by chance. Typically these regions are on the protein surface. The paper found that the microexons code for short amino acid motifs which typically interact with other proteins and ligands. 3 – 27 nucleotides lets you only code for 1 – 9 amino acids.

One well known example of a short interaction motif is RGD (for Arginine Glycine Aspartic acid in the single letter amino acid code). The sequence is found in a family of surface proteins (the integrins) with at least 26 known members. These 3 amino acids are all that is needed for the interns to bind to a variety of extracellular molecules — collagen, fibrin, glycosaminoglycans, proteoglycans. So a 3 amino acid sequence on the surface of a protein can do quite a bit.

Among a set of analyzed neural specific exons (e. g. they were only spliced that way in the brain) found in known disordered regions of the parent protein, 1/3 promoted or disrupted interactions with partner proteins. So regulated exon splicing might specify tissue and cell type specific protein interaction networks (Translation: they might explain why tissues look different even when they express the same genes). The authors regard microExon inclusion/exclusion as protein surface microsurgery.

The paper has found HUNDREDS of evolutionarily highly conserved microexons from RNA-Seq data sets (http://en.wikipedia.org/wiki/RNA-Seq) in various species. Many of them impact neurogenesis and brain function. Regulation of microExons changes significantly during neuronal differentiation. Although microexons represent only 1% of the alternate splice sites seen, they constitute ‘up to’ 1/3 of all evolutionarily conserved neural-regulated alternative splicing between man and mouse.

The inclusion in the final transcript of most identified neural microExons is regulated by a brain specific factor nSR100 (neural specific SR related protein of 100 kiloDaltons)/SRRM4 which binds to intronic enhancer UGC motifs close to the 3′ splice sites, resulting in their inclusion. They are ‘enhanced’ by tissue specific RBFox proteins. nSR100 is reduced in Autism Spectrum DIsorder (really? all? some?). nSR100 is strongly coexpressed in the developing human brain in a gene network module M2 which is enriched for rare de novo ASD assciated mutations.

MicroExons are enriched for lengths which are multiples of 3 nucleotides (implying strong selection pressure to preserve reading frames). The microExons are also enriched in charged amino acids. Most microExons show high inclusion at late stages of neuronal differentiation in genes associated with axon and synapse function. A neural specific microExon in Protrudin/Zfyve27 increases its interaction with Vesicle Associated membrane protein associated Protein VAP) and to promote neurite outgrowth. A 6 nucleotide neural microExon in Apbb1/Fe65 promotes an interaction with Kat5/Tip60. Apbb1 is an adaptor protein functioning in neurite outgrowth.

So inclusion/exclusion of microExons can alter the interactions of proteins involved in neurogenesis. Misregulation of neural specific microexons has been found in autism spectrum disorder (what hasn’t? Pardon the cynicism).

Protein interaction domains haven’t been studied to nearly the extent they need to be, and we know far less about them than we should. All the large molecular machines of the cell (ribosome, mediator, spliceosome, mitochondrial respiratory chain) involve large numbers of proteins interacting with each other not by the covalent bonds beloved by organic chemists, but by much weaker forces (van der Waals,charge attraction, hydrophobic entropic forces etc. etc.).

Designing drugs to interfere (or promote) such interactions will be tricky, yet they should have profound effects on cellular and organismal physiology. Off target effects are almost certain to occur (particularly since we know so little about the partners of a given motif). Showing how potentially useful such a drug can be, a small molecule inhibitor of the interaction of the AIDs virus capsid protein with two cellular proteins (CPSF6, TNPO3) it must interact with to get into the nucleus has been developed. (Unfortunately I’ve lost the reference)

My cousin married a high school dropout a few years ago. Not to worry — he dropped out of high school to go to college, and has a PhD in Electrical Engineering from Berkeley and has worked at Bell labs. He was very interested in combining his math and modeling skills with my knowledge of neurology to make some models of CNS function. I demurred, as I thought we knew too little about the brain to come up with models (which I generally distrust anyway). The basic problem was that I felt we didn’t know all the players in the brain and how they fit together.

MicroExons show this in spades.

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