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.

Here’s a drug target for schizophrenia and other psychiatric diseases

All agree that any drug getting schizophrenics back to normal would be a blockbuster. The more we study its genetics and biochemistry the harder the task becomes. Here’s one target — neuregulin1, one variant of which is strongly associated with schizophrenia (in Iceland).

Now that we know that neuregulin1 is a potential target, why should discovering a drug to treat schizophrenia be so hard? The gene stretches over 1.2 megaBases and the protein contains some 640 amino acids. Cells make some 30 different isoforms by alternative splicing of the gene. Since the gene is so large one would expect to find a lot of single nucleotide polymorphisms (SNPs) in the gene. Here’s some SNP background.

Our genome has 3.2 gigaBases of DNA. With sequencing being what it is, each position has a standard nucleotide at each position (one of A, T, G, or C). If 5% of the population have any one of the other 3 at this position you have a SNP. By 2004 some 7 MILLION SNPs had been found and mapped to the human genome.

Well it’s 10 years later, and a mere 23,094 SNPs have been found in the neuregulin gene, of which 40 have been associated with schizophrenia. Unfortunately most of them aren’t in regions of the gene which code for amino acids (which is to be expected as 640 * 3 = 1920 nucleotides are all you need for coding out of the 1,200,000 nucleotides making up the gene). These SNPs probably alter the amount of the protein expressed but as of now very little is known (even whether they increase or decrease neuregulin1 protein levels).

An excellent review of Neuregulin1 and schizophrenia is available [ Neuron vol. 83 pp. 27 – 49 ’14 ] You’ll need a fairly substantial background in neuroanatomy, neuroembryology, molecular biology, neurophysiology to understand all of it. Included are some fascinating (but probably incomprehensible to the medicinal chemist) material on the different neurophyiologic abnormalities associated with different SNPs in the gene.

Here are a few of the high points (or depressing points for drug discovery) of the review. Neuregulin1 is a member of a 6 gene family, all fairly similar and most expressed in the brain. All of them have multiple splicing isoforms, so drug selectivity between them will be tricky. Also SNPs associated with increased risk of schizophrenia have been found in family members numbers 2, 3 and 6 as well, so neuregulin1 not be the actual target you want to hit.

It gets worse. The neuregulins bind to a family of receptors (the ERBBs) having 4 members. Tending to confirm the utility of the neuregulins as a drug target is the fact that SNPs in the ERBBs are also associated with schizophrenia. So which isoform of which neuregulin binding to which iso form of which ERBB is the real target? Knowledge isn’t always power.

A large part of the paper is concerned with the function of the neuregulins in embryonic development of the brain, leading the the rather depressing thought that the schizophrenic never had a change, having an abnormal brain to begin with. A drug to reverse such problems seems only a hope.

The neuregulin/EBBB system is only one of many genes which have been linked to schizophrenia. So it looks like a post of a 4 years ago on Schizophrenia is largely correct — https://luysii.wordpress.com/2010/04/25/tolstoy-was-right-about-hereditary-diseases-imagine-that/

Happy hunting. It’s a horrible disease and well worth the effort. We’re just beginning to find out how complex it really is. Hopefully we’ll luck out, as we did with the phenothiazines, the first useful antipsychotics.