Research Topic: alternative pre-mRNA splicing

Alternative pre-mRNA splicing allows one gene to produce multiple mRNA isoforms of different structures and coding. It is the major contributor to transcriptome diversity. Tissue-specific alternative splicing is most prevalent in the mammalian brains. The selections of neural specific alternative splice sites are often determined during neuronal differentiation by conserved splicing regulation. Disruption of the splicing regulatory processes has been found in schizophrenia, autism spectrum disorders, Amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and multiple neuromuscular dystrophy. How and why does a neuron choose particular exons? How do these signature exons contribute to the neuronal phenotypes and the establishment of neural circuits? How is neuron specific splicing dynamically regulated in health diseases?

Regulated splicing is most common through the action of RNA binding proteins that recognize sequence elements in the pre-mRNA and subsequently affect splice site choices. The discovery of these splicing factors is still ongoing and their roles in the brain are largely unclear. Our lab is currently focused on a paralog pair of the polypyrimidine tract binding protein PTBP1 and PTBP2. We and others have found that as neural progenitors differentiate, PTBP1 is down-regulated and PTBP2 is induced. Depletion of PTBP1 by RNAi was later reported to sufficiently transdifferentiate fibroblasts to neurons in vitro, indicating that PTBP1 is a master regulator of neuronal cell lineage. PTBP2 (also named nPTB and brPTB) was previously thought to be the neuronal homolog of PTBP1 that contained different activity and function. We recently found that PTBP2 expression also decreases as neurons mature. Now we know that the down-regulation of both PTBPs is necessary for synapse maturation, in part because PTBPs repress a neuronal exon of Psd-95 leading to inhibition of PSD-95 expression.

A synapse is the basic structure where neurons connect and communicate. PSD-95 is a key scaffold protein of excitatory post-synaptic density and promotes synapse maturation. Using CLIP-Seq, we found that both PTBP1 and PTBP2 bind to conserved intronic sequences upstream of Psd-95 exon 18 in embryonic brain.

Introns, once considered junk DNA, are usually not conserved. Conserved introns are more often found flanking regulatory alternative exons, indicating functional regulatory elements utilized by multiple species. The conservation of Psd-95 intron 17 indicates that exon 18 is under evolutionarily conserved regulatory process.

The conserved Psd-95 intron 17 suggests functional significance of its splicing regulation. Indeed, the expression of PSD-95 is probably mostly regulated by splicing of exon 18. Widely considered a neuronal gene since its discovery, Psd-95 is actually transcribed in almost all non-neuronal cells, including human embryonic stem cells (ESC). Thanks to high PTBP1 expression in non-neuronal cells, exon 18 is largely skipped. As ESCs differentiate into neural progenitor cells (NPC) and eventually neurons, exon 18 inclusion increases due to loss of PTBP.

Skipping exon 18 leads to nonsense-mediated mRNA decay (NMD) of the Psd-95 transcripts without productive translation. Thus even though non-neuronal cells are transcribing Psd-95, they don’t produce PSD-95 proteins. Mature neurons express exon 18 and PSD-95 proteins thanks to reduced expression of PTBPs

This would be a classic example of how a neuron specific exon is regulated during development to control expression of an essential synaptic protein and synaptogenesis. The case of Psd-95 also demonstrates a fundamental principle that genetic message is not definitely established right after transcription; instead, it is the splicing pattern that determines the nature of the final mRNA product.

PSD-95 is a brain specific protein. The control of its tissue-specific expression was not clear. Now we believe that splicing regulation of exon 18 enforces neuronal specific expression of PSD-95 proteins. We found that Psd-95 is obviously transcribed in all non-neuronal cells that we examined including embryonic stem cells. In these cells, PTBP1 binds to the intronic sequence upstream of exon 18 and represses exon 18 splicing. Thus, the absence of PSD-95 protein in non-neuronal cells is due to abundant expression of PTBP1 exclusively in non-neuronal cells.

PSD-95 expression is controlled in neuronal plasticity and diseases. To comprehensively dissect the regulation of Psd-95 via exon 18 splicing, one would need to know all the regulators of exon 18 splicing. However, globally identifying regulators of a particular exon had been a challenge. We developed a high-throughput screening strategy using two complementary dual-fluorescence minigene reporters that can be applied to almost any cassette exon to discover its regulators.

Satisfyingly, the method found new RNA binding proteins in addition to PTBPs that affected Psd-95 exon 18 splicing. By studying these modulators, we aim to systemically understand how PSD-95 expression is regulated via exon 18 splicing in both physiological and pathological conditions.