Alternative splicing in the nervous system: an emerging source of diversity and regulation

NEUROSCIENCE PERSPECTIVES Alternative Splicing in the Nervous System: An Emerging Source of Diversity and Regulation Christopher J. Lee and Kris Irizarry Alternative splicing is emerging as a major mechanism of functional regulation in the human genome. Previously considered to be an unusual event, it has been detected by many genomics studies in 40%– 60% of human genes. Moreover, it appears to be of central importance for neuronal genes and other genes involved in “information processing” functions. In this review, we will summarize alternative splicing’s effects on mRNA transcripts, protein products, biological function, and human disease, focusing on genes of neuropsychiatric interest. We will also describe the latest experimental methods and database resources that can help neuroscientists make use of alternative splicing in their own research. Biol Psychiatry 2003;54:771–776 © 2003 Society of Biological Psychiatry Key Words: Alternative splicing, mRNA, genomics, receptors

Introduction

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ost biologists have had the experience of running a gel or blot hoping to see a single band, and of instead seeing multiple bands. Since these “extra” bands are usually of uncertain origin and meaning (perhaps impurities or degradation products), their appearance can seem like an unwelcome surprise. Given the obvious pressure to concentrate on the simplest part of a complicated problem, researchers sometimes focus on the form they were expecting, and leave the “mystery” bands to others. Similarly, the phenomenon of alternative splicing, discovered over 20 years ago, has sometimes escaped notice. Widely regarded as a less common form of functional regulation, it has been much less studied than other mechanisms of regulation such as transcriptional control.

From the Molecular Biology Institute, UCLA Center for Bioinformatics, Center for Genomics and Proteomics, Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California. Address reprint requests to Dr. Christopher J. Lee, Boyer Hall, University of California, Los Angeles, Los Angeles, CA 90095. Received November 15, 2002; revised March 18, 2003; accepted March 24, 2003.

© 2003 Society of Biological Psychiatry

A consensus estimate, found in many recent textbook editions, suggested that alternative splicing occurred in only 5%–15% of genes in complex genomes. Whereas the study of transcription factors and the detailed mechanisms of transcriptional regulation is a vast literature, detailed mechanistic studies of splice regulation have been performed for only a small number of genes (Grabowski and Black 2001; Smith and Valcarcel 2000). Recently, however, genome-wide analyses of mRNA sequence fragments’ (called expressed sequence tags, or ESTs) alignment to the draft human genome sequence, indicated that 40%– 60% of human genes have alternative splice forms (see Modrek and Lee 2002 for a review). At the same time, much progress has been made in identifying and characterizing the functional importance of alternative splice forms in neuronal genes (Grabowski and Black 2001). For all these reasons, now is an excellent time for neuroscientists to learn how alternative splicing might have great importance for their research.

How Alternative Splicing Modifies Transcripts and Proteins Alternative splicing can change the mRNA product in several ways (Figure 1). At its simplest level, an exon can be added or removed (exon skip), or an exon can be selectively lengthened or shortened (alternative 5⬘ or 3⬘ splicing). We will not describe the molecular machinery of mRNA processing, which has been reviewed extensively elsewhere (Maniatis and Tanis 2002; Smith and Valcarcel 2000). A given gene may have a single alternative splicing location, or it may have multiple alternative splice events in different locations. These events can occur independently of each other, or may be coupled in specific combinations. For example, two adjacent exons may be alternatively spliced so that either one or the other is included in the product (mutually exclusive exons). Indeed, this pattern can be expanded to selection of a single exon from a large cassette of exons (one-of-N). By using two or more different promoters in a gene, additional transcript variants differing at their 5⬘ ends can be created (alternative initiation). Equally well, the presence of two 0006-3223/03/$30.00 doi:10.1016/S0006-3223(03)00375-5

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Figure 1. Types of alternative splicing. Regions that are added or removed by alternative splicing are shaded.

or more poly-adenylation sites within a gene can give rise to transcripts with different 3⬘ ends (alternative termination). Exon skipping is most common, followed by alternative 5⬘ and 3⬘ splicing (Modrek et al 2001); mutually exclusive exons are less common. Alternative termination has been observed in 24% of genes (Consortium 2001). Also, in some cases a given splice may not occur, resulting in retention of intronic sequence that would ordinarily be spliced out (intron retention; rare). Even more exotic alterations include RNA editing (e.g., the Q/R site in

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glutamate receptor subunit B), and inteins—alternative protein forms generated by splicing of the polypeptide chain itself (Chevalier and Stoddard 2001). All this complexity can impact protein in ways ranging from subtle to obvious. First, the alternative splice may be in an untranslated region (UTR), and could alter the stability or localization of the mature mRNA without changing the sequence of the translated protein. Alternative initiation often changes the protein’s N-terminus, while alternative termination often modifies the C-terminus. Exon skip typically removes a protein functional domain or motif. Since human internal exons average 145 nt but can range from very small (e.g., 21 nucleotides (nt) up to about 300 nt (Consortium 2001), this can remove just a few amino acids (aa), or a small protein domain (50 –100 aa). Somewhat less frequently, alternative splicing can add a protein domain, with similar size limitations. In cases where two exons are alternatively spliced in a mutually exclusive manner, one protein domain can be replaced by another sequence. While the two domain sequences can be entirely different, sometimes there is only a subtle change. For example, a brain-specific alternative splice form of CDC42 replaces exon V with exon VI. Both encode virtually the same amino acid sequence, but the

Figure 2. Examples of alternative splicing in the nervous system. (A) CDC42. A brain-specific C-terminus introduces a subtle change, removal of a key dilysine binding site motif. (B) DSCAM. Combinatorial splicing of one-of-N cassettes for exons 4, 6, 9 and 17 can theoretically generate 38,016 distinct protein isoforms. (C) mGluR1 receptor: alternative splicing of localization signals in the protein’s C-terminus switches the receptor from axonal targeting to dendritic targeting. (D) tau: mutations that increase splicing of exon 10 add an extra microtubule binding site, and cause filamentous tau aggregates implicated in several neurodegenerative disorders.

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brain-specific form removes two lysine residues that are required for an important binding site (Xu et al 2002) (see Figure 2a). It should be strongly emphasized that alternative splicing can truncate the normal protein product by causing a frameshift (if the added/removed exon sequence is not a multiple of three nucleotides). In some cases this may simply remove a functional domain from the protein’s C-terminus; however, if the new translation STOP site is located too far upstream from the original STOP site (at least 50 nt upstream from an exon-exon junction) this may trigger nonsensemediated decay (NMD) (Lykke-Anderson 2001). This increases degradation of the transcript, reducing the total amount of protein product made. Triggering of NMD by alternative splicing is an area that needs further study.

Alternative Splicing in Information Processing and the Nervous System Genome-wide analyses indicate that alternative splicing is associated with many “information processing” activities in cell biology and in the nervous system. Analyzing the functions of alternatively spliced genes, the majority were found to transmit and regulate signals (e.g., genes for receptors, secreted signals, signal transduction proteins, transcription factors, splicing regulators) rather than to perform metabolic or synthetic functions (Figure 3a) (Modrek et al 2001). That is, most alternatively spliced genes are “white collar” workers who transduce and process information, rather than “blue collar” workers who actually produce energy or real things. Consistent with this result, the main systemic functions identifiable among alternatively spliced genes were neuronal and immunespecific genes (Figure 3b). Indeed, for mammals the function and regulation of alternative splicing has been most extensively studied for neuronal splice forms (see Grabowski and Black 2001). Other data support the strong association of alternative splicing and nervous system functions. Genomics studies of alternative splicing tissue specificity found that the largest group of tissue-specific alternative splice forms was detected in brain, retina, and nerve-derived tissue sources (Figure 3c) (Xu et al 2002). Such systematic statistical studies are reinforced by the anecdotal observation that the literature on neuronal alternative splicing appears to be exploding. For example, on PubMed (October 2002) the query “neuronal alternative splicing” produced 627 publications (most relevant, and most recent), including 57 review articles! This is promising, because while genomics studies have identified many new splice forms, only careful, specific experiments can test whether these forms have any function. We will illustrate alternative splicing’s role in neuronal function with several examples.

Figure 3. Impact of alternative splicing in the human genome. (A) Categorization of alternatively spliced genes by their role in cellular pathways. (B) Categorization by systemic function. (C) Results of a genome-wide analysis of tissue-specificity of alternative splice forms from EST data, showing the number of alternative splice forms that were found to be specific to a single tissue.

Some Examples in Neuronal Functions Receptor Modulation by Alternative Splicing Many different classes of receptors are alternatively spliced in neurons. Serotonin receptors, dopamine receptors, glutamate receptors, and GABA receptors all exhibit alternative splicing with important functional consequences. Many subtypes of serotonin receptor have been reported to be alternatively spliced, including 5HT4 and 5HT7 (Kilpatrick et al 1999), 5HT2, and 5HT3. For

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example, splice variants of 5HT4 modify its C-terminus; those with a shorter C-terminus have increased activity in response to agonist (Claeysen et al 1999). One common theme is the use of alternative splicing to control localization of receptors. The NMDA R1 receptor gene, for example, contains an alternatively spliced exon, the C1 cassette exon. When C1 is included in the transcript, the resulting protein is localized to the cell surface membrane; if C1 is skipped, the protein remains in the cytoplasm. Moreover, it appears C1 drives localization by encoding protein sequence motifs that bind to neurofilaments and also to calmodulin (see Grabowski and Black 2001 for a review). NMDA R2 also has splice variants, which appear to be developmentally regulated (Jelitai et al 2002). The metabotropic glutamate receptor mGluR1 similarly displays a pattern of alternative splice variants that alter its cytoplasmic C-terminal tail and direct its localization (Figure 2b) (Francesconi and Duvoisin 2002). The splice variants mGluR1a and mGluR1b differ in their localization, constitutive activities, and strength of response to agonist. mGluR1b has a short C-terminal tail containing a tripeptide signal (RRK 877–9) that directs it to neuronal axons. mGluR1a has a longer C-terminal tail that contains a dominant signal (residues 1012–71) directing it to dendritic regions of neurons.

Modulation of Neuronal Signaling Alternative splicing plays an important role in modulating many neuronal signaling processes. For example, the ␣1N N-type calcium channel, which controls neurotransmitter release, is alternatively spliced in a way that powerfully alters the channel’s kinetics. Addition of a two amino acid insertion by alternative splicing causes the channel to open almost two times more slowly (Lin et al 1999). Alternative splicing also plays a role in modulating synaptic connections. The cell adhesion molecule apCAM, which facilitates synapse formation and synaptic plasticity in Aplysia, is spliced to make two different isoforms: a membrane spanning isoform and a GPI-linked isoform. It has been shown that the membrane-linked isoform appears to lead to strengthened synaptic connections, while the GPIlinked isoform appears to weaken synaptic connections (Schacher et al 2000).

“Molecular Zipcodes” for Axonal Guidance One extreme example of alternative splicing is provided by the Drosophila Dscam gene (a homologue of the human Down syndrome cell adhesion molecule), which contains four “one-of-N” alternative splicing cassettes (Figure 2c). The alternatively spliced exons each encode immunoglobulin half-domains that are joined combinatorially to create a receptor with many different possible

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affinities—theoretically, multiplying the possible variants from each of these cassettes would yield 38,016 different proteins (Schmucker et al 2000). Dscam appears to act as an axonal guidance receptor, and one interpretation of its function is as a “molecular zipcode” which can encode many possible axonal addresses in a compact way; however, its mammalian homologs lack this combinatorial splicing, highlighting the fact that alternative splicing need not be the same in different genomes. Other axonal guidance molecules such as semaphorins, and genes involved in synapse formation such as agrin and neurexins have also been shown to be alternatively spliced (Grabowski and Black 2001).

Roles in Drug Response and Human Disease Alternative splicing can alter the drug responses of receptors. In the GABAA receptor, for example, splicing generates two variants of the ␥2 subunit (␥2S and ␥2L), changing the receptor’s response to benzodiazepine agonists. Eliminating the ␥2L isoform in a knockout mouse construct alters both the receptor’s ligand affinity in vitro and behavioral responses to the drug in vivo (Quinlan et al 2000). Alternative splicing of bovine norepinephrine transporter has been shown both to alter the rate of norepinephrine uptake and to enhance binding of nisoxetine, a norepinephrine reuptake inhibitor (Kitayama et al 2002). Alternative splicing can also be a hidden factor in human disease processes. One case that has been studied extensively is the role of alternative splice forms of tau proteins in neurodegenerative disorders (Figure 2d) (Buee et al 2000). Six alternative splice forms of tau have been identified, and altered regulation of these splice forms is observed in several diseases. First, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17) was mapped to the tau gene on chromosome 17, and linked to mutations in tau that alter its splicing to increase inclusion of exon 10 (Wilhelmsen 1999). Even synonymous mutations such as L284L (which doesn’t change the protein sequence) can cause this effect, providing striking evidence that the disease process is triggered at the level of transcript processing, not protein. Such mutations can double the rate of inclusion of exon 10 in tau transcripts, by weakening a putative splicing repressor element within exon 10 (D’Souza and Schellenberg 2000). Inclusion of exon 10 adds an extra microtubule binding site to the tau protein, and appears to result in filamentous tau aggregates characteristic of FTDP-17 pathology. Similar tau aggregates with alteredsplice form ratios are observed in alzheimer’s disease and other neurodegenerative disorders, prompting their proposed grouping as “tauopathies.”

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Table 1. Database Resources for Alternative Splicing Database

Organism

Content

AltExtron

Human

ASAP

Human; mouse; others in progress

AsMamDB

Human, mouse, rat

Ensembl

Human; mouse, zebrafish; fugu; mosquito

Intronerator

C. elegans

Mouse Genome Informatics

Mouse

NCBI RefSeq

Many

PubMed

Many

SwissProt

Many

TAP

Human

796 alternatively spliced genes, including exon and splice site data (Clark and Thanaraj 2002). www.ebi.ac.uk/asd/altextron 32,000 alternative splice relationships in human genes (Lee et al 2003); 12,000 in mouse. Gene structures, tissue specificity. www.bioinformatics.ucla.edu/ASAP 1,563 alternatively spliced genes, including their alternative splicing patterns, structures, chromosomal locations, products, and tissue data (Ji et al 2001). 166.111.30.65/ASMAMDB.html Experimental and predicted gene structures, mapping to genome. www.ensembl.org Introns and alternative splicing in C. elegans (Kent and Zahler 2000). www.cse.ucsc.edu/⬃kent/intronerator 1,257 alternative splice forms in mouse genes (Mangan and Frazer 1999). ftp.informatics.jax.org/pub/reports/PRB_AltTranscripts.rpt Human curated mRNA sequences, mapping to genome. www.ncbi.nlm.nih.gov/LocusLink/refseq.html Because there is not yet a truly unified database of alternative splicing data, in many cases reading literature is the only way to find splice forms for a gene. www.ncbi.nlm.nih.gov Human curated annotations of alternative splice forms drawn from published literature. www.expasy.org 365 alternatively spliced human genes, based on RefSeq (Kan et al 2001). sapiens.wustl.edu/⬃zkan/TAP

ASAP, Alternative Splicing Annotation Project; ASMamDB, Alternative Splicing Mammalian Database; NCBI, National Center for Biotechnology Information; TAP, Transcription Assembly Project.

Tools for Analyzing Alternative Splicing The availability of complete genome sequences has revolutionized the study of alternative splice forms. Previously, the observation of “variant transcripts” or protein isoforms—those pesky extra bands on your gels and blots— could not be translated easily or confidently to a specific alternative splicing event. In the absence of the genomic sequence and exon/intron structure of your gene, it was not possible to check whether the variants could be genuine alternative splicing, by matching against the possible exon sequences of the gene, checking for valid splice sites, etc. Similarly, in the absence of a complete genome sequence, it was hard to know whether the variants might actually be paralogs of your gene— highly similar, but encoded by an altogether different gene. Having the genome sequence makes it possible to check all these possibilities rapidly (provided you scrutinize the sequence data carefully). This fact is creating an explosion of alternative splice form discovery throughout many areas of biomedical research, as people acquire the tools for identifying, verifying and characterizing specific splice forms in their genes of interest. This data may eventually help researchers solve one major difficulty in alternative splicing research—the lack of genetic approaches for testing the function of splice variants.

A second major factor is the avalanche of alternative splice form discovery from high-throughput experimental methods such as EST sequencing (Modrek and Lee 2002). In the human genome alone, over 30,000 alternative splicing relationships have been identified (Xu et al 2002), and the number is constantly growing with new EST sequencing. To make use of all this, you first need tools for identifying possible splice forms of interest to you, and second, tools for designing new experiments to probe and characterize these forms. Table 1 lists several online database resources that provide this type of information for a number of different organisms. Ideally, you need to see the detailed gene structure, all possible splice forms, and their impact on the protein. Seeing which exon sequences and exon-exon junctions are unique to a given form is essential for designing specific probes, and for predicting the molecular weight differences to expect on your gels or reverse transcription polymerase chain reaction (RT-PCR). Experimental methodology for studying alternative splice forms is in rapid flux. Traditional methods such as Northern or Western blots are now complemented by methods like RT-PCR that can probe the specific difference between one transcript and another. Whereas se-

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quencing (e.g., ESTs) is required for identifying entirely new forms, hybridization methods are most useful for quantifying previously known forms. Taking this idea to its logical extreme, a number of groups are endeavoring to build “splicing arrays”—DNA microarrays that can distinguish specific alternative splice forms en masse (Clark et al 2002; Hu et al 2001; Yeakley et al 2002). Eventually, these technologies should enable simultaneous detection of many splice forms throughout the entire human genome, in each mRNA sample that is analyzed. Perhaps, in the future, you will never have to ignore an “extra band” again!

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