Cell-specific Alternative Splicing Research Articles

Disruption of Cdh23 exon 68 splicing leads to progressive hearing loss in mice by affecting tip-link stability

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.

Prospects of Identifying Alternative Splicing Events from Single-Cell RNA Sequencing Data

Background: The advent of single-cell RNA sequencing (scRNA-seq) technology has offered unprecedented opportunities to unravel cellular heterogeneity and functions. Yet, despite its success in unraveling gene expression heterogeneity, accurately identifying and interpreting alternative splicing events from scRNA-seq data remains a formidable challenge. With advancing technology and algorithmic innovations, the prospect of accurately identifying alternative splicing events from scRNA-seq data is becoming increasingly promising Objective: This perspective aims to uncover the intricacies of splicing at the single-cell level and their potential implications for health and disease. It seeks to harness scRNA-seq's transformative power in revealing cell-specific alternative splicing dynamics and aims to propel our understanding of gene regulation within individual cells to new heights. Methods: The perspective grounds its method on recent literature along with the experimental protocols of single-cell RNA-seq and methods to identify and quantify the alternative splicing events from scRNA-seq data. Results: This perspective outlines the promising potential, challenges, and methodologies for leveraging different scRNA-seq technologies to identify and study alternative splicing events, with a focus on advancing our understanding of gene regulation at the single-cell level. Conclusion: This perspective explores the prospects of utilizing scRNA-seq data to identify and study alternative splicing events, highlighting their potential, challenges, methodologies, biological insights, and future directions.

Cell-type specific regulator RBPMS switches alternative splicing via higher-order oligomerization and heterotypic interactions with other splicing regulators.

Alternative pre-mRNA splicing decisions are regulated by RNA binding proteins (RBPs) that can activate or repress regulated splice sites. Repressive RBPs typically harness multivalent interactions to bind stably to target RNAs. Multivalency can be achieved by homomeric oligomerization and heteromeric interactions with other RBPs, often mediated by intrinsically disordered regions (IDRs), and by possessing multiple RNA binding domains. Cell-specific splicing decisions often involve the action of widely expressed RBPs, which are able to bind multivalently around target exons, but without effect in the absence of a cell-specific regulator. To address how cell-specific regulators can collaborate with constitutive RBPs in alternative splicing regulation, we used the smooth-muscle specific regulator RBPMS. Recombinant RBPMS is sufficient to confer smooth muscle cell specific alternative splicing of Tpm1 exon 3 in cell-free assays by preventing assembly of ATP-dependent splicing complexes. This activity depends upon a C-terminal IDR that facilitates dynamic higher-order self-assembly, cooperative binding to multivalent RNA and interactions with widely expressed splicing co-regulators, including MBNL1 and RBFOX2, allowing cooperative assembly of stable cell-specific regulatory complexes.

Epigenetic Connection of the Calcitonin Gene-Related Peptide and Its Potential in Migraine.

The calcitonin gene-related peptide (CGRP) is implicated in the pathogenesis of several pain-related syndromes, including migraine. Targeting CGRP and its receptor by their antagonists and antibodies was a breakthrough in migraine therapy, but the need to improve efficacy and limit the side effects of these drugs justify further studies on the regulation of CGRP in migraine. The expression of the CGRP encoding gene, CALCA, is modulated by epigenetic modifications, including the DNA methylation, histone modification, and effects of micro RNAs (miRNAs), circular RNAs, and long-coding RNAs (lncRNAs). On the other hand, CGRP can change the epigenetic profile of neuronal and glial cells. The promoter of the CALCA gene has two CpG islands that may be specifically methylated in migraine patients. DNA methylation and lncRNAs were shown to play a role in the cell-specific alternative splicing of the CALCA primary transcript. CGRP may be involved in changes in neural cytoarchitecture that are controlled by histone deacetylase 6 (HDAC6) and can be related to migraine. Inhibition of HDAC6 results in reduced cortical-spreading depression and a blockade of the CGRP receptor. CGRP levels are associated with the expression of several miRNAs in plasma, making them useful peripheral markers of migraine. The fundamental role of CGRP in inflammatory pain transmission may be epigenetically regulated. In conclusion, epigenetic connections of CGRP should be further explored for efficient and safe antimigraine therapy.

BaseScope™ Approach to Visualize Alternative Splice Variants in Tissue.

Defining the cell-specific alternative splicing landscape in complex tissues is an important goal to gain functional insights. Deep-sequencing techniques coupled to genetic strategies for cell identification has provided important cues on cell-specific exon usage in complex tissues like the nervous system. BaseScope™ has emerged as a powerful and highly sensitive alternative to in situ hybridization to determine exon composition in tissue with spatial and morphological context. In this protocol, we will review how BaseScope was utilized to detect the e37a-Cacna1b splice variant of the presynaptic calcium channel CaV2.2 or N-type. This splice variant arises from a pair of mutually exclusive exons (e37a and e37b). E37a-Cacna1b is heavily underrepresented relative to e37b-Cacna1b and both exons share 60% of their sequence. By using BaseScope™, we were able to discover that e37a-Cacna1b is expressed in excitatory pyramidal neurons of hippocampus and cortex, as well as motor neurons of the ventral horn of the spinal cord.

Compound heterozygous c.598_612del and c.1746-20C\u2009>\u2009G CAPN3 genotype cause autosomal recessive limb-girdle muscular dystrophy-1: a case report

BackgroundAutosomal recessive limb–girdle muscular dystrophy-1 (LGMDR1), also known as calpainopathy, is a genetically heterogeneous disorder characterised by progression of muscle weakness. Homozygous or compound heterozygous variants in the CAPN3 gene are known genetic causes of this condition. The aim of this study was to confirm the molecular consequences of the CAPN3 variant NG_008660.1(NM_000070.3):c.1746-20C > G of an individual with suspected LGMDR1 by extensive complementary DNA (cDNA) analysis.Case presentationIn the present study, we report on a male with proximal muscular weakness in his lower limbs. Compound heterozygous NM_000070.3:c.598_612del and NG_008660.1(NM_000070.3):c.1746-20C > G genotype was detected on the CAPN3 gene by targeted next-generation sequencing (NGS). To confirm the pathogenicity of the variant c.1746-20C > G, we conducted genetic analysis based on Sanger sequencing of the proband’s cDNA sample. The results revealed that this splicing variant disrupts the original 3′ splice site on intron 13, thus leading to the skipping of the DNA fragment involving exon 14 and possibly exon 15. However, the lack of exon 15 in the CAPN3 isoforms present in a blood sample was explained by cell-specific alternative splicing rather than an aberrant splicing mechanism. In silico the c.1746-20C > G splicing variant consequently resulted in frameshift and formation of a premature termination codon (NP_000061.1:p.(Glu582Aspfs*62)).ConclusionsBased on the results of our study and the literature we reviewed, both c.598_612del and c.1746-20C > G variants are pathogenic and together cause LGMDR1. Therefore, extensive mRNA and/or cDNA analysis of splicing variants is critical to understand the pathogenesis of the disease.

Cell-specific exon methylation and CTCF binding in neurons regulate calcium ion channel splicing and function.

Cell-specific alternative splicing modulates myriad cell functions and is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line. In vivo, hypomethylation of an alternative exon specifically in nociceptors, likely permits CTCF binding and expression of CaV2.2 channel isoforms with increased opioid sensitivity in mice. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states - and reveal a potential target for the treatment of chronic pain.

Cacna1b alternative splicing impacts excitatory neurotransmission and is linked to behavioral responses to aversive stimuli

Presynaptic CaV2.2 channels control calcium entry that triggers neurotransmitter release at both central and peripheral synapses. The Cacna1b gene encodes the α1-pore forming subunit of CaV2.2 channels. Distinct subsets of splice variants of CaV2.2 derived from cell-specific alternative splicing of the Cacna1b pre-mRNA are expressed in specific subpopulations of neurons. Four cell-specific sites of alternative splicing in Cacna1b that alter CaV2.2 channel function have been described in detail: three cassette exons (e18a, e24a, and e31a) and a pair of mutually exclusive exons (e37a/e37b). Cacna1b mRNAs containing e37a are highly enriched in a subpopulation of nociceptors where they influence nociception and morphine analgesia. E37a-Cacna1b mRNAs are also expressed in brain, but their cell-specific expression in this part of the nervous system, their functional consequences in central synapses and their role on complex behavior have not been studied. In this report, we show that e37a-Cacna1b mRNAs are expressed in excitatory projection neurons where CaV2.2 channels are known to influence transmitter release at excitatory inputs from entorhinal cortex (EC) to dentate gyrus (DG). By comparing behaviors of WT mice to those that only express e37b-CaV2.2 channels, we found evidence that e37a-CaV2.2 enhances behavioral responses to aversive stimuli. Our results suggest that alternative splicing of Cacna1b e37a influences excitatory transmitter release and couples to complex behaviors.

One Gene, Many Proteins: Mapping Cell-Specific Alternative Splicing in Plants

Pre-mRNA alternative splicing (AS) generates protein variants from a single gene that can create novel regulatory opportunities. In this issue of Developmental Cell, Li etal. (2016) present a high-resolution expression map of AS events in Arabidopsis root tissues, giving insight into cell-type- and stage-specific AS mechanisms in plants.

Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians.

In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans.

Regulation of Tissue-Specific Alternative Splicing: C. elegans as a Model System.

Alternative pre-mRNA splicing serves as an elegant mechanism for generating transcriptomic and proteomic diversity between cell and tissue types. In this chapter, we highlight key concepts and outstanding goals in studies of tissue and cell-specific alternative splicing. We place particular emphasis on the use of C. elegans as a tractable model organism for in vivo studies of alternative splicing between tissues and also at single cell resolution. We describe our current understanding of tissue and cell-specific regulation in the animal, and emerging techniques that will allow for future mechanistic studies as well as systems level investigations of spatio-temporal splicing under laboratory conditions and in response to environmental stimuli.

Reversion to an Embryonic Alternative Splicing Program Enhances Leukemia Stem Cell Self-Renewal

BackgroundFormative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established.MethodsWhole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013).ResultsWe performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance.ConclusionIn summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature.

Alternative pre-mRNA splicing is a highly cell type-specific process essential to generating protein diversity. However, the mechanisms responsible for the establishment and maintenance of heritable cell-specific alternative-splicing programs are poorly understood. Recent observations point to a role of histone modifications in the regulation of alternative splicing. Here we report a new mechanism of chromatin-mediated splicing control involving a long noncoding RNA (lncRNA). We have identified an evolutionarily conserved nuclear antisense lncRNA, generated from within the human FGFR2 locus, that promotes epithelial-specific alternative splicing of FGFR2. The lncRNA acts through recruitment of Polycomb-group proteins and the histone demethylase KDM2a to create a chromatin environment that impairs binding of a repressive chromatin-splicing adaptor complex important for mesenchymal-specific splicing. Our results uncover a new function for lncRNAs in the establishment and maintenance of cell-specific alternative splicing via modulation of chromatin signatures.

Cell-Specific Alternative Splicing of Drosophila Dscam2 Is Crucial for Proper Neuronal Wiring

How a finite number of genes specify a seemingly infinite number of neuronal connections is a central question in neurobiology. Alternative splicing has been proposed to increase proteome diversity in the brain. Here we show that cell-specific alternative splicing of a cell-surface protein is crucial for neuronal wiring. Down syndrome cell adhesion molecule 2 (Dscam2) is a conserved homophilic binding protein that can induce repulsion between opposing neurons. In the fly visual system, L1 and L2 neurons both require Dscam2 repulsion, but paradoxically, they also physically contact each other. We found that the cell-specific expression of two biochemically distinct alternative isoforms of Dscam2 prevents these cells from repelling each other. Phenotypes were observed in the axon terminals of L1 and L2 when they expressed the incorrect isoform, demonstrating a requirement for distinct isoforms. We conclude that cell-specific alternative splicing is a mechanism for achieving proper connectivity between neurons.

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.

Differential Ubiquitination and Proteasome Regulation of CaV2.2 N-Type Channel Splice Isoforms

Ca(V)2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca(V)2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca(V)2 channels. Here we show that Ca(V)2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca(V)2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca(V)2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a--not e37b--is selected during alternative splicing of Ca(V)2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca(V)2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca(V)2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing.

RBM4 down-regulates PTB and antagonizes its activity in muscle cell–specific alternative splicing

Alternative splicing contributes largely to cell differentiation and functional specification. We previously reported that the RNA-binding protein RBM4 antagonizes the activity of splicing factor PTB to modulate muscle cell-specific exon selection of α-tropomyosin. Here we show that down-regulation of PTB and its neuronal analogue nPTB during muscle cell differentiation may involve alternative splicing-coupled nonsense-mediated mRNA decay. RBM4 regulates PTB/nPTB expression by activating exon skipping of their transcripts during myogenesis. Moreover, RBM4 and PTB target a common set of transcripts that undergo muscle cell-specific alternative splicing. Overexpression of RBM4 invariably promoted expression of muscle cell-specific isoforms, which recapitulated in vivo alternative splicing changes during muscle differentiation, whereas PTB acted oppositely to RBM4 in expression of mRNA isoforms specific for late-stage differentiation. Therefore, RBM4 may synergize its effect on muscle cell-specific alternative splicing by down-regulating PTB expression and antagonizing the activity of PTB in exon selection, which highlights a hierarchical role for RBM4 in a splicing cascade that regulates myogenesis.

Fox-3 and PSF interact to activate neural cell-specific alternative splicing

Fox-1 family (Fox) proteins, which consist of Fox-1 (A2BP1), Fox-2 (Rbm9) and Fox-3 (NeuN) in mammals, bind to the RNA element UGCAUG and regulate alternative pre-mRNA splicing. However the mechanisms for Fox-regulated splicing are largely unknown. We analyzed the expression pattern of the three Fox proteins as well as neural cell-specific alternative splicing of a cassette exon N30 of nonmuscle myosin heavy chain (NMHC) II-B in the mouse central nervous system. Histological and biochemical analyses following fluorescence-activated cell sorting demonstrate a positive correlation of N30 inclusion and Fox-3 expression. Further, we identified polypyrimidine tract binding protein-associated splicing factor (PSF) as an interacting protein with Fox-3 by affinity-chromatography. In cultured cells, enhancement of N30 inclusion by Fox-3 depends on the presence of PSF. PSF enhances N30 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Fox-3 is recruited to the UGCAUG element downstream of N30 in the endogenous NMHC II-B transcript in a PSF-dependent manner. This study is the first to identify PSF as a coactivator of Fox proteins and provides evidence that the Fox-3 and PSF interaction is an integral part of the mechanism by which Fox proteins regulate activation of alternative exons via a downstream intronic enhancer.

Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing

Alternative pre-mRNA splicing predominates in the nervous systems of complex organisms including humans dramatically expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type CaV2.2 calcium channels in nociceptors. Using an exon replacement strategy in mice, we show that mutually exclusive splicing in the CaV2.2 gene modulates N-type channel function in nociceptors leading to a change in morphine analgesia. Exon 37a enhances μ-opioid receptor mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a spinal morphine analgesia is weakened in vivo without influencing the basal response to noxious thermal stimuli. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.