Alternative splicing and nonsense-mediated mRNA decay enforce neural specific gene expression

Abstract

Alternative pre-mRNA splicing is a fundamental regulatory process for most mammalian multi-exon genes to increase proteome diversity. Nonsense-mediated mRNA decay (NMD) is a conserved mRNA surveillance mechanism to mitigate deleterious effects caused by gene mutations or transcriptional errors. Coupling alternative splicing and NMD (AS-NMD), in which alternative splicing switches between translational and NMD isoforms, results in fine-tuning overall gene expression to, in turn, expand the functional activities of these two post-transcriptional regulatory processes. AS-NMD is known for maintaining homeostatic expression of many RNA-binding proteins. We further show that AS-NMD is a conserved mechanism among mammals to induce developmental expression of the synaptic scaffold protein PSD-95. Comparing gene sequences between human Psd-95 and its ancestral orthologues indicates that AS-NMD regulation of mammalian Psd-95 is a product of selective pressure and that it enforces neural-specific expression of PSD-95 proteins in mammals. Invertebrate homolog of Psd-95 is not subjected to AS-NMD regulation and its protein product does not exhibit neural-specific expression. Given the prevalence of alternative splicing regulation in the mammalian nervous system, neural-specific expression of many other genes could be controlled by AS-NMD in a similar manner. We discuss the implication of these discoveries, as well as the challenges in generalizing the regulation and functional activity of AS-NMD.

Figure 1

Psd-95 exon 18 is neural-specific in human cells. Exon 17 to Exon 20 of PSD-95 transcript is shown. PTBP1 binds to the Psd-95 pre-mRNA transcripts in human embryonic stem cells (ESC). PTBP1 iCLIP-Seq tags are enriched in conserved intronic sequences upstream of Psd-95 exon 18. As a result, exon 18 is mostly skipped in ESC. Exon 18 becomes more included as ESCs differentiate into neural progenitor cells (NPC) and eventually neurons.

Figure 2

Intron 17 contributes to neuron-specific expression of PSD-95 protein in mammals. (A) Sequence alignment of human, rhesus, mouse and playtypus Psd-95 and its ancestral orthologue D. melanogaster Dlg1. Introns are lowercase. Exons are uppercase and highlighted by the green blocks corresponding to human exon 17, 18 and 19 sequentially. Only the intron-exon and exon-intron junction sequences are shown. Nucleotides between the shown junctions are represented by dots. A gap in D. melanogaster Dlg1 is represented by dashes. Sequences are segmented by the vertical lines in the lower panel to calculate sequence identity between platypus and human or fruit fly for each segment. (B) Therefore, Dlg1, the “ancestral” orthologue of Psd-95, does not have an intron homologous to mammalian intron 17. Dlg1 is expressed in many cells, including epithelial cells, as shown by. Mammalian Psd-95 has intron 17 whose splicing is regulated by PTBP1 and PTBP2. As a result, PSD-95 expression is restricted to the brain. The image of PSD-95 expression is from GENESAT.

Figure 3

Sequential expression of PTBP1 and PTBP2 during development is conserved between mouse and human in various brain regions. (A) Temporal expression patterns of PTBP1 and PTBP2 in developing mouse neocortices (Zheng et al., 2012). PTBP1/2 protein expression is first normalized to internal control GAPDH. The normalized time-serial expression is then normalized to the expression level at E16 for PTBP1 and E12 for PTBP2. (B) Temporal expression patterns of PTBP1 and PTBP2 in various human brain regions at 8, 9, 12, 13, 16, 17, 19,21, 24, 25,26, 35, 37 pcw (post- conception week), 4 months, 10 months, as well as 1, 2, 3, 4, 8, 11, 13, 15, 18, 19, 21, 23, 30, 36, and 37 years of age. Each color block represents one specific age.