Why drug discovery is so hard (particularly in the brain): Reason #28: The brain processes its introns very differently

Useful drug discovery for neurologic and psychiatric disease is nearly at a standstill. It isn’t for want of trying by basic researchers and big and small pharma. A recent excellent review [ Neuron vol. 87 pp. 14 – 27 ’15 ] helps explain why. In short, the brain processes its protein coding genes rather differently.

This post assumes you know what introns, exons and alternate splicing are. For pretty much all the needed background see the following.

First: https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/
Second:https://luysii.wordpress.com/2010/07/11/molecular-biology-survival-guide-for-chemists-ii-what-dna-is-transcribed-into/

When splicing first came out I started making a list of proteins which were alternatively spliced. It is now safe to assume that any gene containing introns (95% of all protein coding genes [ Proc. Natl. Acad. Sci. vol. 112 pp. 17985 – 17990 ’08 ]) results in several protein products due to alternative splicing. The products produced vary from tissue to tissue, probably because most tissues express different splicing regulators.

Here are a few. A2BP1 (aka Rbfox1, aka FOX1) is a brain specific RNA splicing factor found only in postmitotic terminally differentiated neurons. It is deleted in 10% of glioblastomas. Another is nSR100 (neural Specific Related protein of 100 kiloDaltons) — see later.

To show how crucial alternative splicing is for the every existence of the brain, consider this. The neuronal splicing regulator PTBP2 is barely expressed in most tissues. It is upregulated in neurons. Both PTBP1 and PTBP2 are repressors of neural alternative splicing (but some genes are actually enhanced). In a given region of the brain either PTPB1 or PTBP2 is expressed (but not both). PTBP1 promotes skiping of a neural specific exon (exon #10) in PTBP2 transcripts. This exposes a premature termination codon in PTBP2 leading to nonsense mediated decay (NMD). PTPB1 is expressed in most nonNeural tissues and neural precursor cells, but is silenced in developing neurons by the microRNA miR-124. The mRNA for PTBP2 contains an alternative exon which triggers nonsense mediated decay (NMD) when skipped. Inclusion of the exon requires positive transacting factors such as nSR100 in neurons. Repression is mediated by PTBP1 in undifferentiation. microRNAs (which ones?) downregulate PTBP1 during neuronal differentiation, relieving the negative regulation of PTBP2. Depletion of PTBP1 in fibroblasts is enough for PTBP2 induction and neuronal transdifferentiation.

It gets more complicated still. PTBP1 inhibits splicing of introns at the 3′ end of some genes involved in presynaptic function. This results in nuclear retention and turnover via components of the nuclear RNA surveillance machinery. As PTBP1 is downregulated during neuronal differentiation, the target introns are spliced out and the mature mRNAs are found.

Now we get to microExons, something unknown until 2014. For more details see — https://luysii.wordpress.com/2015/01/04/microexons-great-new-drugable-targets/.
Briefly, 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.

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 said to be 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. Recall that every 3 nucleotides in mRNA codes for an amino acid. This implies strong selection pressure was used to preserve reading frames as 3n+1 and 3n+2 produce a frameshift. The microExons are enriched in charged amino acids. Most microExons show high inclusion at late stages of neuronal differentiation in genes associated with axon formation and synapse function. A neural specific microExon in Protrudin/Zfyve27 increases its interation with Vessicale Associated membrane protein associated Protein VAP) and to promote neurite outgrowth.

[ Proc. Natl. Acad. Sci. vol. 112 pp. 3445 – 3450 ’15 ] Deep mRNA sequencing of mouse cerebral cortex expanded the list of alternative splicing events TENfold and showed that 72% of multiexon genes express multiple splice variants. Among the newly discovered alternatively spliced exon are 1,104 exons involved in nonsense mediated decay (NMD). THey are enriched in RNA binding proteins including splicing factors. Another set of alternatively spliced NMD exons is found in genes coding for chromatin regulators. Conservation of NMD exons is found in lower vertebrates, but those involving chromatin regulators are found later into the mammalian lineage. So the transcriptome in the brain is even more complicated.

A bit more about the actual effects on protein structure of alternate splicing. The sites chosen for this aren’t random. Cell and tissue differentially regulated alternative splicing events are significantly UNDERrepresented in functionally defined folded domains in proteins, they are enriched in regions of protein disorder that typically are surface accessible and embed short linear interaction motifs (with other proteins and ligands). Among a set of analyzed neural specific exons enriched in disordered regions, 1/3 promoted or disrupted interactions with partner proteins. So regulated exon splicing might specify tissue and cell type specific protein interaction networks. They regard their inclusion/exclusion as protein surface microsurgery.

How much can a little microexon do to protein function? Here’s an example of a 6 nucleotide microexon (two amino acids). Insertion of the microExon in the nuclear adaptor protein Apbb1 enhances its interaction with Kat5/Tip60 a histone deacetylase. The microExon adds Arginine and Glutamic acid to a phosphotyrosine binding domain (PTB domain) which binds Kat4. This enhances binding.

Had enough? The complexity is staggering and I haven’t even talked about recursive splicing — that’s for another post, but here’s a reference if you can’t wait — [ Nature vol. 521 pp. 300 – 301, 371 – 375, 376 – 379 ’15 ]. Pity the drug chemist figuring out which alternatively spliced form of a brain protein to attack (particularly if it hasn’t been studied for microExons).