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Chapter 9 Alternative mRNA splicing in the nervous system
J . Joosse, R . M . Buijs and F.J.H. Tilders (Eds.)
Progress in Brain Reseorch, Vol. 92
0 1992 Elsevier Science Publisher5 B . V . All rlghfs reserved
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CHAPTER 9
Alternative mRNA splicing in the nervous system Julian F. Burke, Kerris E. Bright, Elaine Kellett, Paul R. Benjamin and Susan E. Saunders Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Fulmer, Brighton, BNI 9QG, U.K .
Introduction Peptide neurotransmitters were classically obtained by purification from large quantities of tissue on the basis of a specific bioassay. Once purified, sequenced and synthesized the localization and physiological actions could then be explored. However, the application of molecular biological techniques has resulted in a substantial increase in our knowledge of not only the structure of neuropeptides but also of the precursors from which they are derived and the genes from which the precursors are transcribed. From such studies it has emerged that the genes encoding neuropeptides are invariably complex structures and often occur in families within the genome, for example mammals have evolved a family of opioid peptide genes (Hollt, 1985) and molluscs such as Lymnaea and Aplysia have well-characterized gene families encoding egg laying hormones (ELH) (Scheller et al., 1982) and insulin-related peptides (Smit et al., 1988), respectively. Furthermore, in Lymnaea the FMRFamide-related peptides are encoded in multiple copies in at least two exons (Linacre et al., 1990; Saunders et al., 1991). How such neuropeptide genes are regulated to ensure the specific tem-
Correspondence: J.F. Burke, Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falrner, Brighton, BNl 9QG, U.K. Fax: (44) (273) 678433.
poral and spatial expression required for the correct functioning of the nervous system has become one of the major questions facing molecular neurobiologists. Eukaryotic gene expression requires the activities of complex biochemical machinery to transcribe, process and transport mature mRNA before it can be translated to yield functional products. Each of the activities involved in the pathway of protein production is a potential point at which the expression of a gene can be regulated. In this chapter the factors which regulate the synthesis of mRNA and in particular the splicing of neuropeptide precursor mRNAs will be discussed.
The initiation of messenger RNA (mRNA) synthesis The expression of neuropeptide genes is cell- or tissue-specific, often developmentally regulated and may be subject to influence from both external (e.g. synaptic or hormonal input) and internal cues. A primary control point at which such cues act is the initiation of mRNA synthesis; cells respond by turning genes on or off and by modulating the extent of transcription of active genes (Mitchell and Tjian, 1989). Although the mechanisms and biochemical pathways by which cells integrate physiological cues to bring about appropriate transcriptional changes are still largely unknown, it is
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clear that the frequency of initiation of mRNA synthesis depends ultimately on factors that interact with specific sequence elements in gene promoters; such factors are denoted as trans-acting factors and the sequence elements as cis-acting elements respectively (for reviews see Dynan and Tjian, 1985; Jones et al., 1988; Mermelstein et al., 1989). Systematic in vitro mutagenesis and DNAmediated gene transfer studies have identified two distinct types of cis-acting regulatory sequences: promoters, which are located close t o the initiation site and act in a position-dependent fashion, and enhancers which can be located far from the initiation site and act in a position and orientation independent manner (for reviews see Serfling et al., 1985; Ptashne, 1986).
Processing and splicing of pre-mRNA The product of transcription, termed the premRNA, heterogenous nuclear RNA (hnRNA) or the primary transcript, undergoes a series of nuclear processing events before a mature messenger RNA (mRNA) is produced. In additon to 5 ’ capping with 7-methyl guanosine (Salditt-Georgieff et al., 1980) and the addition of a poly(A) tail to the 3’ end of nascent transcripts (Birnstiel et al., 1985), the non-coding intervening sequences (introns) must be removed and the exons joined together. The splicing of introns has to occur with great precision and fidelity, requiring the correct identification of splice sites and precise ligation of exons to avoid disruption of the open reading frame (ORF). The mechanisms that exist whereby cells are able to accomplish this task have been the subject of many recent reviews (e.g. Green, 1986;
Padgett et al., 1986; Aebi and Weissmann, 1987; Maniatis et al., 1987; Sharp, 1987).
Mechanisms of constitutive splicing Analysis of eukaryotic genes has revealed conserved sequences at both the 5 ‘ donor and 3 ‘ acceptor site (Mount, 1982). The 5’ splice-site consensus is (C/A)AG/GURAGU (where R = purine, and the intron - exon boundary is denoted by the line between G / G ) and the 3 ‘ splice-site consensus sequence is YAG/G (where Y = pyrimidine). Additional conserved sequences include a loosely defined branch point sequence, YNYURAY, usually located about 30 nucleotides upstream from the 3 ’ splice site and a stretch of pyrimidines juxtaposing the 3 ’ splice site. Although the consensus sequences are important they are not sufficient to define the site used in vivo since similar sequences are found in positions other than the actual splice sites (Smith et al., 1989). A general view of splicing envisages interactions between trans-acting factors such as the snRNPs with the template and with each other to create a molecular architecture within which the splicing process can occur (Guthrie and Patterson, 1988). The interactions of cis-elements with the transacting factors during the formation of spliceosomes are necessary not only for constitutive splicing in which each and every exon is joined in an orderly fashion, but also for “alternative” splicing (processing, where functional splice sites that are selected in some circumstances are completely bypassed by the splicing machinery in others; Smith et al., 1989). Alternative pre-mRNA splicing ap-
Fig. 1. A. Illustration of the differential expression of the bovine pre-protachykinin A (PPT-A) gene. The primary transcript is alternatively processed to either a-PPTmRNA by the cassette mode of splicing; exon 6 representing the cassette. The primary translation product is cleaved at the sites denoted * to produce the combination of peptides shown. The “potential” peptides have yet to be characterized. B. Illustration of the alternative RNA processing pathways involved in the expression of the calcitonin gene. The primary transcript is alternatively processed by differential cleavage/polyadenylation sites (denoted A) to produce either calcitonin or calcitonin gene-related peptide (CGRP) encoding mRNAs. The primary translation products are cleaved at the sites denoted * to produce the peptides shown. (CCP denotes a 16 amino acid COOH-terminal calcitonin cleavage product). The N-terminal (Nterm) and C-terminal (C-term) peptides have yet to be characterized.
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pears to be a widespread device for regulating gene expression and generating isoform diversity (Leff et al., 1986; Breitbart et al., 1987) particularly in the nervous system where some 30% of the genes known to be predominantly expressed in this tissue employ mechanisms of alternative splicing (Sutcliffe and Milner, 1988). Tissue-specific alternative RNA splicing The functional consequence of most cases of alternative splicing is the production of protein isoforms sharing extensive regions of identity and varying only in specific domains. The activities of protein isoforms can thus be subtly varied by alternative splicing. For example, the Drosophila Shaker locus which encodes the major structural component of the A type K f channel, generates diverse transcripts via alternative splicing which code for at least nine different proteins that vary within the N- and C-terminal regions (Schwartz and Costa, 1986; Sutcliffe and Milner, 1988). Injection of two different Shaker mRNAs into Xenopus oocytes demonstrated that the different channels have distinct inactivation kinetics (Timpe et al., 1988). Regulated alternative RNA processing can also be used to specify the localization of proteins as illustrated by the neural cell adhesion molecules (NCAMs) which use alternative splicing to generate various membrane-bound and secreted forms (Cower et al., 1988). Alternative splicing can act as a simple on/off switch or produce products which have no activity as in the case of the Drosophila P element transposase gene which when transcribed in somatic cells produces a product with a retained intron; the resultant protein being non-functional (Laski et al., 1986). Many genes produce transcripts with heterogeneity within the 5 ' and 3' untranslated regions. These sequences can regulate translational efficiency (Kozak, 1986) and RNA stability (Brawerman, 1987; Raghow, 1987). Alternative processing involving these regions thus provides the potential for regulating levels of particular mRNAs and translation products.
Genes that produce a pre-mRNA encoding multiple potential peptides within discrete exons can use alternative splicing to generate different repertoires of peptides in different cells/tissues. Several neuropeptide/hormone genes have been shown to employ this strategy to generate diversity from their transcription units. The tachykinins, so named due to their ability to rapidly induce the contraction of gut tissue, are a family of structurally related neuropeptides all sharing a common C-terminal amino acid sequence (Erspramer, 1981). Three members of this family have been characterized in mammalian systems; substance P, substance K (neurokinin A), and neuromedin K. Substance P and substance K are encoded within the same gene, the pre-protachykinin A gene (PPT-A) which has been cloned in both bovine and rat (Nawa et al., 1984; Krause et al., 1987) whereas neuromedin K is encoded by a separate gene (the PPT-B gene) (Kotani et al., 1986; Bonner et al., 1987). The PPT-A gene consists of 7 exons in which part of exon 3 specifies substance P and exon 6 encodes the sequence containing substance K (Fig. 1A). Krause et al., have demonstrated by cDNA cloning and nuclease protection experiments that 3 different rat mRNAs ( a , /3 and y PPTmRNA) are derived by alternative RNA splicing. The P-PPTmRNA contains all 7 exons, y-PPTmRNA excludes exon 4 from its structure and a-PPTmRNA excludes exon 6. Thus both /3- and y-PPTmRNA encode substance P and substance K, whereas a-PPTmRNA encodes substance P but not substance K. The mRNA corresponding to the rat y-PPTmRNA has yet to be conclusively identified in bovine but the a- and PPPTmRNAs which have the same coding potential as their rat homologues, are expressed. Furthermore, quantitative analysis of the rat PPT mRNAs indicates that y-PPTmRNA > P-PPTmRNA > > a-PPTmRNA at a ratio of approximately 80:20:1 (Carter and Krause, 1990) and that this ratio exists in all rat tissues where the gene is expressed; thus indicating that the splicing mechanism is not regulated in a tissue-specific manner in the adult rat. However, the bovine PPT-A gene
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was shown to be differentially spliced in a tissuespecific manner (Nawa et al., 1984). The bovine aPPTmRNA was found to be more abundant in CNS tissue compared to /3-PPTmRNA (3: 1); whereas 6-PPTmRNA was more abundant than aPPTmRNA in thyroid and intestine tissues. The tachykinin PPT-A gene is not unique in its use of differential exon expression as a mechanism of tissue-specific gene regulation. The gene encoding calcitonin (CT) and calcitonin gene-related peptide (CGRP), as shown from studies of both the rat and human gene, also employs this strategy (Amara et al., 1982; Rosenfeld et al., 1983; Amara et al., 1984; Jonas et al., 1985). The gene is composed of 6 exons (Fig. 1B). Exons 1 - 3 are constitutive and contain the 5’ untranslated and common coding regions. Exon 4 contains the CT coding sequence and a poly(A) addition site and is used in thyroid C cells. In neural tissue exon 3 is spliced instead to exons 5 and 6, which contain the CGRP coding and 3 ’ untranslated sequences and poly(A) addition site. CT functions as a circulating calcium homeostatic hormone (Fontaine et al., 1987) while CGRP appears to have more diverse actions having both neuromodulatory and trophic activities (Rosenfeld, 1983). In addition to coding for CT or CGRP both messages encode two other putative peptides, the functions of which have yet to be elucidated.
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Neuron-specific alternative RNA splicing Both the calcitonin/CGRP gene and the PPT-A gene employ mechanisms of alternative RNA processing to differentially express mRNAs in a tissuespecific manner, i.e. the alternative mRNAs are expressed in either neural or non-neural tissue. However, the R15 neuropeptide precursor of the marine slug Aplysia generates two different mRNAs by a mechanism of alternative splicing which results in their differential expression within a single tissue, the nervous system (Buck et al., 1987). The two different mRNAs differ in a single region whereby a 6-nucleotide sequence present in one mRNA (R15-1) is replaced by a different 48-
Fig. 2. In situ hybridization showing expression of FMRFamide exon (A) and GDPFLRFamide exon (B) specific sequences in two alternate sections from the Lymnaea brain. In most instances the same cell is visible in both sections. The DNA hybridization probes and procedures are as described in Linacre et al., (1990) and Saunders et al., (1991). ( x 120.)
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an RNA transcript within the Aplysia nervous system can result in differential mRNA expression within individual neurons (Buck et al., 1987). In the freshwater snail Lyrnnaea, the FMRFamide (Phe-Met-Arg-Phe-NH2) related peptides are encoded by two distinct exons (Linacre et al., 1990; Saunders et al., 1991). The question arises as to where these sequences are expressed in the brain. It is possible that both are expressed in the same cells or alternatively each exon could be expressed in a different cell, i.e. expression is exclusive. By the use of in situ hybridization
nucleotide sequence in the alternative mRNA (R15-2). The 48-nucleotide sequence was shown by genomic mapping to be encoded within a single exon and encodes a portion of the a 1 peptide, a peptide isolated from neuron R15 possessing potent osmoregulatory activity. Thus the 48 for 6 nucleotide substitution is likely to be of physiological significance. Many neurons within the abdominal ganglion express the R15 gene, as shown by in situ hybridization, but expression of the 48-nucleotide exon appears to be specific to the R15 neuron (Buck et al., 1987). Thus alternative processing of
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6 x SDPFLRFa 1 x SDPYLRFa 1 x SDPFFRFa 1 x EFFPLa Fig. 3. Model for expression of the FMRFamide gene in Lymnaea. Expression of the FMRFamide-related peptide gene appears to be regulated by alternative splicing of a primary transcript by the "mutually exclusive exon" mode of splicing. In this case the expression is regulated in a neuron-specific manner. The gene consists of two peptide encoding exons encoding the tetrapeptides and heptapeptides. Also shown is a hydrophobic leader sequence (HLl) encoded by another exon which is spliced onto both peptide-encoding exons. It is not clear whether the second hydrophobic leader sequence (R), in-frame with the FMRFamide exon, is expressed. The mature peptides and the number of copies of each peptide sequence are shown. The data for this model is derived from Linacre et al., (1990), Saunders et al., (1991) and Saunders et al. (in press).
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with either FMRFamide (tetrapeptide) or GDPFLRFamide (heptapeptide) specific DNA probes, the expression pattern in individual neurons can be determined. Fig. 2 shows alternate sections through the CNS of Lymnaea hybridized to FMRFamide specific sequences (Linacre et al., 1990) or GDPFLRFamide-specific exon sequences (Saunders et al., 1991). It can be seen that cells expressing the FMRFamide exon in the cytoplasm (as judged by the “halo” pattern of hybridization) do not express the GDPFLRFamide exon in the cytoplasm, and vice versa. The FMRFamide-gene has been shown to consist of at least 3 exons (Linacre et al., 1990; Saunders et a]., 1991; Saunders et al., 1992) and appears to produce at least 2 different mature transcripts by a “mutually exclusive” mode of alternative splicing where either the tetrapeptide or heptapeptide - exon (Fig. 3) is spliced into a specific mRNA, exclusion or inclusion of both simultaneously does not occur (Fig. 3; Saunders et al., 1992). However, until the gene has been fully characterized at both the 5’ and 3’ termini the utilization of other mechanisms of alternative RNA processing by this gene, such as differential polyadenylation site usage, cannot be entirely ruled out. In comparison to the tachykinin and calcitonin genes, the spliced transcripts derived from the Lymnaea FMRFamide-related peptide gene do not appear to be differentially expressed in a tissue-specific manner but in a neuron-specific manner; like the expression of the Aplysia R15 precursor encoding gene which has been shown to be regulated in a neuron-specific manner (Buck et al., 1987). Individual neurons within the Lymnaea nervous system also regulate the processing of an RNA transcript from a single gene in different ways (Fig. 2 and Saunders et al., 1992). However, the expression of the FMRFamide-related peptide gene is the clearest example of neuron-specific gene expression shown to date and occurs in a more diverse range of behaviourally related neurons.
Possible mechanisms of regulation of neuronspecific alternative RNA splicing Evidence to date strongly suggests that the Lymnaea FMRFamide-related peptide gene, as is being discovered for an increasing number of genes, employs the strategy of alternative RNA processing to generate diversity from its transcription unit (Fig. 3 and Saunders et al. in press). However, the mechanisms involved in the regulation of alternative processing pathways are presently little understood. Signals must reside in information encoded within the gene transcript (cis-elements), but may require additional control by diffusible (transacting) factors. Cases of alternative splicing where different mRNAs derived from structurally homogeneous primary transcripts are expressed in a cell type- or developmental stage-specific manner, clearly demonstrate that the splicing “environment” of different cells varies for these transcripts. The differences in “environment” are likely to be determined by variability in type or concentration of trans-acting factors. The nature of such factors is for most cases unclear, however all the components of the spliceosome including the snRNAs and other components of the snRNPs are potential candidates. The action of trans-acting factors is likely to affect the folding of the premRNA to present particular donor or acceptor pairs in proximity, or inhibit or promote the usage of a particular splicing junction. In vitro studies have suggested that the secondary structure adopted by RNA molecules can influence splicesite selection, and thus determine the outcome of the splicing event. For example, sequences in the introns at either side of exon 6 in the rat tachykinin PPT-A gene have been proposed to promote intrastrand annealing, forming a hairpin structure which could prevent inclusion of this exon, thus resulting in the formation of a-PPTmRNA. Similarly, sequences adjacent to exon 4 could potentially interact to exclude this exon, thus producing y-
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PPTmRNA (Carter and Krause, 1990). Such a hypothesis is based on the finding that splice sites sequestered in genetically engineered RNA hairpin loops can be omitted in the splicing process (Solnick, 1985; Eperon et al., 1986). However, although such observations established the potential of secondary structure in influencing splicing choices, they did not establish whether such a mechanism is normally used in vivo. It has subsequently been shown that when such constructs were introduced into HeLa cells the hairpin loops had little or no affect on the splicing choices (Solnick and Lee, 1987). These results suggest that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all (possibly due to the actions of endogenous RNA helicases) (Solnick and Lee, 1987). The elements involved in regulating the tissuespecific expression of the rat calcitonin gene have been the focus of several studies. Interestingly, it has been shown that the tissue-specific processing is the result of alternative splice-site selection primarily regulated by cis-active sequences at the calcitonin-specific 3 ’ splice junction, and not controlled by differential polyadenylation site usage (Emerson et al., 1989). The results obtained from this study are consistent with a model in which cisactive sequences serve to inhibit the formation of calcitonin mRNA in CGRP-producing cells, most probably by binding trans-acting factors that are specific to CGRP-producing cells. The nature of such a putative factor(s) has yet to be elucidated. Analysis of the mechanisms operating to regulate the differential processing of the FMRFamiderelated peptide gene transcript will obviously await the complete characterization of the gene. However, from the results shown here (and Saunders et al., 1992) it would appear that different neurons within the CNS offer a different splicing “environment” to the primary transcript. It is possible that one of the spliced transcripts is produced by a “default mode” of splicing and the other by a “regulated mode”, requiring the expression of specific trans-acting factors within those cells ex-
pressing that transcript, but it is also equally possible that both types of splicing reaction require specific factors. Comparison with other FMRFamide related genes The structure of the Aplysia gene has yet to be fully determined, mainly due to the lack of genomic clones. However, analysis of cDNA clones has suggested that the prohormone precursor is encoded within at least two exons (Taussig and Scheller, 1986). A more detailed analysis of the Drosophila FMRFamide-related peptide gene has revealed that this gene consists of two exons separated by a -2.5 kb intervening sequence (Chin et al., 1990; Schneider and Taghert, 1990). However the prohormone precursor is entirely encoded within the second exon, the first exon encodes an untranslated leader sequence. In comparison, the Lymnaea gene consists of at least 3 exons. The first exon encodes a spliced hydrophobic leader sequence which, although translated, does not appear in the prohormone structures. Furthermore, 2 prohormones can derive from this gene, one encoding the tetrapeptides and the other encoding the heptapeptides, both of which are encoded within discrete exons (Saunders et al., submitted). The splicing strategies used by the genes also appear to differ. For example, sequencing of cDNAs derived from the Aplysia gene suggests that the primary transcript can be spliced to produce mature transcripts which vary in relation to the copy number of FMRFamide encoding sequences contained within them (Taussig and Scheller, 1986). The precise nature of the multiple transcripts which have been detected in Aplysia is unclear but it has been proposed that they are the products of “intra-exonic” splicing events. However, it is also possible that the cDNAs are not true representations of the mRNA transcript(s) but are cloning artifacts which arose as the result of recombination between the highly repeated FMRFamide encoding sequences. In favour of the alternative splicing hypothesis is the detection of multiple transcripts that have different sizes within neural tissue (Schaefer et al.,
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1985). However, analysis of the expression of these putative spliced transcripts has not extended to in situ hybridization analysis and thus differential expression of this gene by alternative splicing has not convincingly been shown. Analysis of cDNAs derived from the Lymnaea gene coupled with the in situ hybridization data presented here suggests that the primary precursor derived from this gene is subject to alternative RNA splicing. However, the splicing mechanism used in this case is “interexonic” rather than “intra-exonic” as is possibly the case in Aplysia and thus results in the production of mature transcripts that differ in the nature of peptides encoded rather than the copy number of one peptide as in Aplysia. In contrast t o the molluscan FMRFamide-related peptide genes described, the Drosophila gene appears to give rise to a single transcript and thus the potential for alternative splicing appears slight (Schneider and Taghert, 1988; Schneider and Taghert, 1990; Chin et al., 1990). In addition t o the organization of the genes, the organization and FMRFamide-related peptide content of the prohormones derived from each gene differs. The Aplysia gene encodes 28 copies of FMRFamide, and a single copy of FLRFamide. The Lymnaea gene also encodes these two peptides but the copy number is different: 9 copies of FMRFamide and 2 copies of FLRFamide are encoded within one exon (Linacre et al., 1990). The other exon encodes the heptapeptides GDPFLRFamide and SDPFLRFamide (Saunders et al., 1991). The Drosophila gene, however, does not encode these forms of tetrapeptides and heptapeptides but encodes a diverse range of related peptides, i.e. 13 hepta- and nonapeptides that represent N-terminal extensions of the FMRFamide sequence, the copy number of each individual peptide sequence varies from 1 - 5. Thus although transcription of the Drosophila gene appears to produce less complex transcripts than either the Aplysia or Lymnaea genes, it encodes a more complex repertoire of FMRFamiderelated peptides. The expression of the Drosophila gene has been analysed by both immunocytochemistry (White et al., 1986; Chin et al., 1990) and in situ hybridiza-
tion (Chin et al., 1990), and has been shown to be expressed in - 500 cells within the nervous system. The possibility that the prohormone undergoes differential processing has been suggested from the study by Chin et al., (1990), whereby an antibody generated against a peptide spanning a potential proteolytic cleavage site within the precursor is shown to stain a unique set of cells. However, the specificity of this antibody to the FMRFamide precursor has yet to be conclusively shown and thus the question of whether differential processing actually occurs remains unanswered. Thus, while the splicing strategy of the Drosophila gene appears simple it may use mechanisms of post-translational processing to generate diversity from its transcriptional unit. Whether the prohormones derived from the Lymnaea gene are also subject to differential processing has yet to be determined. One significant similarity between the expression of the FMRFamide-related peptide encoding genes of Lymnaea and Drosphila is their widespread distribution throughout the nervous system, which suggests important physiological roles for FMRFamide-related peptides in both systems. The axonal projections of the 40 - 50 Drosophila FMRFamide-immunoreactive central neurons has suggested that they are involved in a variety of physiological processes (Chin et al., 1990) and include neurosecretory cells which project to release sites in the periphery and interneurons whose processes are confined to the CNS (Schneider and Taghert, 1990). Our data show that the Lymnaea gene is similarly expressed in a wide variety of cell types. However, unlike the Drosophila CNS which because of its small size has not been extensively studied in terms of neuropeptide physiology, the Lymnaea CNS has proved to be an ideal model system in which to perform studies of peptidergic and non-peptidergic neural systems.
References Aebi, M. and Weissmann, C. (1987) Precision and orderliness in splicing. Trends Genet. 3: 102- 107.
124 Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and Evans, R. (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature, 298: 240 - 244. Amara, S.G., Evans, R.M. and Rosenfeld, M.G. (1984) Calcitonin/CGRP transcription unit: Tissue specific expression involves selective use of alternative polyadenylation sites. Mol. CeN. Biol., 4: 2151 -2160. Birnstiel, M.L., Busslinger, M. and Strub, K. (1985) Transcription termination and 3’ processing: The end is in site. Cell, 41: 349-359. Bonner, T., Affolter, H.U., Young, A.C. and Young, W.S. (1987) A cDNA encoding the precursor of the rat neuropeptide neurokinin B. Mol. Brain. Rex, 2: 243 - 249. Brawerman, G. (1987) Determinants of messenger RNA stability. Cell, 48: 5-6. Breitbart, R.E., Andreadis, A. and Nadal-Ginard, B. (1987) Alternative splicing: A ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annu. Rev. Biochem., 56: 467 -495. Buck, L.B., Bigelow, J.M. and Axel, R. (1987) Alternative splicing in individual Aplysia neurons generates neuropeptide diversity. CeN, 51: 127- 133. Carter, M.S. and Krause, J.E. (1990) Structure, expression and some regulatory mechanisms of the rat preprotachykinin gene encoding Substance P, Neurokinin A, Neuropeptide K, and Neuropeptide y. J . Neurosci., 10: 2203 - 2214. Chin, A.C., Reynolds, E.R. and Scheller, R.H. (1990) Organisation and expression of the Drosophila FMRFamiderelated prohormone gene. DNA Cell. Biol. 9: 263 - 271. Dynan, W.S. and Tjian, R. (1985) Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. Nature, 316: 774-778. Emerson, R.B., Hedjron, F., Yeakley, J.M., Guise, J.W. and Rosenfeld, M.G. (1989) Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor. Nature, 341: 76- 80. Eperon, L.P., Estibeiro, J.P. and Eperon, I.C. (1986) The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA. Nature, 324: 280 - 282. Erspramer, V. (1981) The tachykinin peptide family. Trends. Neurosci., 4: 267 - 269. Fontaine, B., Klarsfield, A. and Changeux, J.P. (1987) Calcitonin gene related peptide and muscle activity regulate acetylcholine receptor CY subunit mRNA levels by distinct intracellular pathways. J . CeN. Biol., 105: 1337- 1342. Cower, H . J . , Barton, G.H., Elson, V.L., Thompson, J., Moore, S.E., Dickson, G . and Walsh, F.S. (1988) Alternative splicing generates a secreted form of N-CAM in muscle and brain. Cell, 55: 955 - 964. Green, M.R. (1986) Pre-mRNA splicing. Annu. Rev. Genet., 20: 671 -708. Guthrie, C . and Patterson, B. (1988) Spliceosomal snRNAs. Annu. Rev. Genet., 22: 387-419.
Hollt, V. (1985) Multiple endogenous opioid pepetides. In D. Bousfield (Ed.), Neurotransmitters in Action. Elsevier, pp. 188- 193. Jonas, V., Lin, C.R., Kawashima, E., Semon, D., Swanson, L.W. Mermod, J.J., Evans, R.M. and Rosenfeld, M.G. (1985) Alternative RNA processing events in human calcitonidcalcitonin gene-related peptide gene expression. Proc. Natl. Acad. Sci. U.S.A., 82: 1994- 1998. Jones, N.C., Rigby P.W.J. and Ziff, E.B. (1988) Transacting protein factors and the regulation of eukaryotic transcripion: lessons from studies on DNA tumor viruses. Genes Dev., 2: 267 281. Kotani, H., Hoshimaru, M., Nawa, H. and Nakanishi, S. (1986) Structure and gene organisation of bovine neuromedin K precursor. Proc. Natl. Acad. Sci. U.S.A., 83: 7074 - 7078. Kozak, M . (1986) Regulation of protein synthesis in virus infected animal cells. Adv. Virus. R e x , 31: 229-292. Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S. and Hershey, A.D. (1987) Three rat preprotachykinin niRNAs encode the neuropeptides substance P and neurokinin A . Proc. Nu//. Acad. Sci. U.S.A., 84: 881 -885. Laski, R.A., Rio, D.C. and Ruben G.M. (1986) Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell, 44: 7 19. Leff, S.E. and Rosenfeld, M.G. (1986) Complex transcriptional units: Diversity in gene expression by alternative RNA processing. Annu. Rev. Biochem. 55: 1091 - 11 17. Linacre, A., Kellett, E., Saunders, S.E., Bright, K., Benjamin, P.R., and Burke, J.F. (1990). Cardioactive neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and novel related peptides are encoded in multiple copies by a single gene in the snail Lymnaea stagnalis. J . Neurosci., 10: 412-419. Maniatis, T., Goodbourn, S. and Fisher, J.A. (1987) Regulation of inducible and tissue-specific gene expression. Science, 236: 1237 - 1244. Mermelstein, F.H., Flores, 0. and Reinberg, D. (1989) Initiation of transcription by RNA polymerase 11. Biochim. Biophys. Acta, 1009: 1 - 10. Mitchell, P . J . and Tjian, R . (1989) Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science, 245: 371 - 378. Mount, S.M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res., 10: 459- 472. Nawa, H., Kotani, H . and Nakanishi, S. (1984) Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature, 3 12: 729 - 734. Padgett, R.A., Grabowski, P.J., Konarska, M.M., Seiler, S. and Sharp, P.A. (1986) Splicing of messenger RNA precursors. Annu. Rev. Biochem. 55: 1119-1150. Ptashne, M. (1986) Gene regulation by proteins acting nearby and at a distance. Nature, 322: 697. Raghow, R. (1987) Determinants of messenger RNA stability. Cell, 48: 5 - 6. Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, -
-
125 L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature, 304: 129- 135. Salditt-Georgieff, M., Harpold, M. and Chen-Kiang, S. (1980) The addition of 5’ cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped. Cell, 19: 69-78. Saunders, S.E., Bright, K.E., Kellett, E., Benjamin, P.R. and Burke, J.F. (1991) Neuropeptides GDPFLRFamide and SDPFLRFamide are encoded by an exon 3’ to FMRFamide in the snail Lymnaea sfagnalis. J . Neurosci., 11: 140- 745. Saunders, S.E., Kellett, E., Bright, K., Benjamin, P.R. and Burke, J.F. (1992) Cell specific alternative RNA splicing of a FMRFamide gene transcript in the brain. J. Neurosci., 12: 1033 - 1039. Schaefer, M., Picciotto, M.R., Kreiner, T., Kaldany, R.R., Taussig, R. and Scheller, R.H. (1985). Aplysia neurons express a gene encoding multiple FMRFamide neuropeptides. Cell, 41: 451-467. Schneider, L.E. and Taghert, P.H. (1988) Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH, (FMRFamide). Proc. Nail. Acad. Sci. U.S.A., 85: 1993 - 1991. Schneider, L.E. and Taghert, P.H. (1990) Organization and expression of the Drosophila Phe-Met-Arg-Phe-NH, neuropeptide gene. J. Biol. Chem., 265: 6890 - 6895. , Schwartz, J.P. and Costa, E. (1986) Hybridization approaches to the study of neuropeptides. Annu. Rev. Neurosci., 9: 221 - 304.
Serfling, E., Jasin, M. and Schaffner, W. (1985)Enhancers and eukaryotic gene expression. Trends Genet., 1: 224. Sharp, P.A (1987) Splicing of messenger RNA precursors. Science, 235: 766 - 111. Smit, A.B., Vreugdenhil, E., Ebberink, R.H.M., Geraerts, W.P.M., Klootwijk, J . and Joosse, J . (1988) Growthcontrolling molluscan neurons produce the precursor of an insulin-related peptide. Nature, 331: 535 - 538. Smith, C.W.J., Pattern, J.K. and Nadal-Ginard, B. (1989) Alternative splicing in the control of gene expression. Annu. Rev. Genet., 23: 527 - 571. Solnick, D. (1986) Alternative splicing caused by RNA secondary structure. Cell, 43: 667 -676. Solnick, D. and Lee, S.T. (1987) Amount of RNA secondary structure required to induce an alternative splice site selection in eukaryotic pre-messenger RNA. Mol. Cell. Biol., 7: 3 194 - 3 198. Sutcliffe, J.G. and Milner, R.J. (1988) Alternative mRNA splicing: The Shaker Gene. Trends Gener., 4: 297 299. Taussig, R. and Scheller, R.H. (1986) The Aplysia FMRFamide gene encodes sequences related to mammaliam brain peptides. DNA, 5: 543 - 561. Timpe, L.C., Schwartz, T.L., Tempel, B.L., Papazion, D.M., Jan, Y.N. and Jan, L.Y. (1988) Expression of functional potassium channels from Shaker cDNA in Xenopus Oocytes. Nature, 331: 143- 145. White, K . , Hurteau, T . and Punsal, P . (1986) NeuropeptideFMRFamide-like immunoreactivity in Drosophila: Development and distribution. J . Comp. Neurol., 247: 430-438.
Note added in Pro03 FMRFamide sequences can be found in GENBANK: M87479.
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Progress in Brain Reseorch, Vol. 92
0 1992 Elsevier Science Publisher5 B . V . All rlghfs reserved
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CHAPTER 9
Alternative mRNA splicing in the nervous system Julian F. Burke, Kerris E. Bright, Elaine Kellett, Paul R. Benjamin and Susan E. Saunders Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Fulmer, Brighton, BNI 9QG, U.K .
Introduction Peptide neurotransmitters were classically obtained by purification from large quantities of tissue on the basis of a specific bioassay. Once purified, sequenced and synthesized the localization and physiological actions could then be explored. However, the application of molecular biological techniques has resulted in a substantial increase in our knowledge of not only the structure of neuropeptides but also of the precursors from which they are derived and the genes from which the precursors are transcribed. From such studies it has emerged that the genes encoding neuropeptides are invariably complex structures and often occur in families within the genome, for example mammals have evolved a family of opioid peptide genes (Hollt, 1985) and molluscs such as Lymnaea and Aplysia have well-characterized gene families encoding egg laying hormones (ELH) (Scheller et al., 1982) and insulin-related peptides (Smit et al., 1988), respectively. Furthermore, in Lymnaea the FMRFamide-related peptides are encoded in multiple copies in at least two exons (Linacre et al., 1990; Saunders et al., 1991). How such neuropeptide genes are regulated to ensure the specific tem-
Correspondence: J.F. Burke, Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falrner, Brighton, BNl 9QG, U.K. Fax: (44) (273) 678433.
poral and spatial expression required for the correct functioning of the nervous system has become one of the major questions facing molecular neurobiologists. Eukaryotic gene expression requires the activities of complex biochemical machinery to transcribe, process and transport mature mRNA before it can be translated to yield functional products. Each of the activities involved in the pathway of protein production is a potential point at which the expression of a gene can be regulated. In this chapter the factors which regulate the synthesis of mRNA and in particular the splicing of neuropeptide precursor mRNAs will be discussed.
The initiation of messenger RNA (mRNA) synthesis The expression of neuropeptide genes is cell- or tissue-specific, often developmentally regulated and may be subject to influence from both external (e.g. synaptic or hormonal input) and internal cues. A primary control point at which such cues act is the initiation of mRNA synthesis; cells respond by turning genes on or off and by modulating the extent of transcription of active genes (Mitchell and Tjian, 1989). Although the mechanisms and biochemical pathways by which cells integrate physiological cues to bring about appropriate transcriptional changes are still largely unknown, it is
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clear that the frequency of initiation of mRNA synthesis depends ultimately on factors that interact with specific sequence elements in gene promoters; such factors are denoted as trans-acting factors and the sequence elements as cis-acting elements respectively (for reviews see Dynan and Tjian, 1985; Jones et al., 1988; Mermelstein et al., 1989). Systematic in vitro mutagenesis and DNAmediated gene transfer studies have identified two distinct types of cis-acting regulatory sequences: promoters, which are located close t o the initiation site and act in a position-dependent fashion, and enhancers which can be located far from the initiation site and act in a position and orientation independent manner (for reviews see Serfling et al., 1985; Ptashne, 1986).
Processing and splicing of pre-mRNA The product of transcription, termed the premRNA, heterogenous nuclear RNA (hnRNA) or the primary transcript, undergoes a series of nuclear processing events before a mature messenger RNA (mRNA) is produced. In additon to 5 ’ capping with 7-methyl guanosine (Salditt-Georgieff et al., 1980) and the addition of a poly(A) tail to the 3’ end of nascent transcripts (Birnstiel et al., 1985), the non-coding intervening sequences (introns) must be removed and the exons joined together. The splicing of introns has to occur with great precision and fidelity, requiring the correct identification of splice sites and precise ligation of exons to avoid disruption of the open reading frame (ORF). The mechanisms that exist whereby cells are able to accomplish this task have been the subject of many recent reviews (e.g. Green, 1986;
Padgett et al., 1986; Aebi and Weissmann, 1987; Maniatis et al., 1987; Sharp, 1987).
Mechanisms of constitutive splicing Analysis of eukaryotic genes has revealed conserved sequences at both the 5 ‘ donor and 3 ‘ acceptor site (Mount, 1982). The 5’ splice-site consensus is (C/A)AG/GURAGU (where R = purine, and the intron - exon boundary is denoted by the line between G / G ) and the 3 ‘ splice-site consensus sequence is YAG/G (where Y = pyrimidine). Additional conserved sequences include a loosely defined branch point sequence, YNYURAY, usually located about 30 nucleotides upstream from the 3 ’ splice site and a stretch of pyrimidines juxtaposing the 3 ’ splice site. Although the consensus sequences are important they are not sufficient to define the site used in vivo since similar sequences are found in positions other than the actual splice sites (Smith et al., 1989). A general view of splicing envisages interactions between trans-acting factors such as the snRNPs with the template and with each other to create a molecular architecture within which the splicing process can occur (Guthrie and Patterson, 1988). The interactions of cis-elements with the transacting factors during the formation of spliceosomes are necessary not only for constitutive splicing in which each and every exon is joined in an orderly fashion, but also for “alternative” splicing (processing, where functional splice sites that are selected in some circumstances are completely bypassed by the splicing machinery in others; Smith et al., 1989). Alternative pre-mRNA splicing ap-
Fig. 1. A. Illustration of the differential expression of the bovine pre-protachykinin A (PPT-A) gene. The primary transcript is alternatively processed to either a-PPTmRNA by the cassette mode of splicing; exon 6 representing the cassette. The primary translation product is cleaved at the sites denoted * to produce the combination of peptides shown. The “potential” peptides have yet to be characterized. B. Illustration of the alternative RNA processing pathways involved in the expression of the calcitonin gene. The primary transcript is alternatively processed by differential cleavage/polyadenylation sites (denoted A) to produce either calcitonin or calcitonin gene-related peptide (CGRP) encoding mRNAs. The primary translation products are cleaved at the sites denoted * to produce the peptides shown. (CCP denotes a 16 amino acid COOH-terminal calcitonin cleavage product). The N-terminal (Nterm) and C-terminal (C-term) peptides have yet to be characterized.
Bovine PPT gene
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pears to be a widespread device for regulating gene expression and generating isoform diversity (Leff et al., 1986; Breitbart et al., 1987) particularly in the nervous system where some 30% of the genes known to be predominantly expressed in this tissue employ mechanisms of alternative splicing (Sutcliffe and Milner, 1988). Tissue-specific alternative RNA splicing The functional consequence of most cases of alternative splicing is the production of protein isoforms sharing extensive regions of identity and varying only in specific domains. The activities of protein isoforms can thus be subtly varied by alternative splicing. For example, the Drosophila Shaker locus which encodes the major structural component of the A type K f channel, generates diverse transcripts via alternative splicing which code for at least nine different proteins that vary within the N- and C-terminal regions (Schwartz and Costa, 1986; Sutcliffe and Milner, 1988). Injection of two different Shaker mRNAs into Xenopus oocytes demonstrated that the different channels have distinct inactivation kinetics (Timpe et al., 1988). Regulated alternative RNA processing can also be used to specify the localization of proteins as illustrated by the neural cell adhesion molecules (NCAMs) which use alternative splicing to generate various membrane-bound and secreted forms (Cower et al., 1988). Alternative splicing can act as a simple on/off switch or produce products which have no activity as in the case of the Drosophila P element transposase gene which when transcribed in somatic cells produces a product with a retained intron; the resultant protein being non-functional (Laski et al., 1986). Many genes produce transcripts with heterogeneity within the 5 ' and 3' untranslated regions. These sequences can regulate translational efficiency (Kozak, 1986) and RNA stability (Brawerman, 1987; Raghow, 1987). Alternative processing involving these regions thus provides the potential for regulating levels of particular mRNAs and translation products.
Genes that produce a pre-mRNA encoding multiple potential peptides within discrete exons can use alternative splicing to generate different repertoires of peptides in different cells/tissues. Several neuropeptide/hormone genes have been shown to employ this strategy to generate diversity from their transcription units. The tachykinins, so named due to their ability to rapidly induce the contraction of gut tissue, are a family of structurally related neuropeptides all sharing a common C-terminal amino acid sequence (Erspramer, 1981). Three members of this family have been characterized in mammalian systems; substance P, substance K (neurokinin A), and neuromedin K. Substance P and substance K are encoded within the same gene, the pre-protachykinin A gene (PPT-A) which has been cloned in both bovine and rat (Nawa et al., 1984; Krause et al., 1987) whereas neuromedin K is encoded by a separate gene (the PPT-B gene) (Kotani et al., 1986; Bonner et al., 1987). The PPT-A gene consists of 7 exons in which part of exon 3 specifies substance P and exon 6 encodes the sequence containing substance K (Fig. 1A). Krause et al., have demonstrated by cDNA cloning and nuclease protection experiments that 3 different rat mRNAs ( a , /3 and y PPTmRNA) are derived by alternative RNA splicing. The P-PPTmRNA contains all 7 exons, y-PPTmRNA excludes exon 4 from its structure and a-PPTmRNA excludes exon 6. Thus both /3- and y-PPTmRNA encode substance P and substance K, whereas a-PPTmRNA encodes substance P but not substance K. The mRNA corresponding to the rat y-PPTmRNA has yet to be conclusively identified in bovine but the a- and PPPTmRNAs which have the same coding potential as their rat homologues, are expressed. Furthermore, quantitative analysis of the rat PPT mRNAs indicates that y-PPTmRNA > P-PPTmRNA > > a-PPTmRNA at a ratio of approximately 80:20:1 (Carter and Krause, 1990) and that this ratio exists in all rat tissues where the gene is expressed; thus indicating that the splicing mechanism is not regulated in a tissue-specific manner in the adult rat. However, the bovine PPT-A gene
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was shown to be differentially spliced in a tissuespecific manner (Nawa et al., 1984). The bovine aPPTmRNA was found to be more abundant in CNS tissue compared to /3-PPTmRNA (3: 1); whereas 6-PPTmRNA was more abundant than aPPTmRNA in thyroid and intestine tissues. The tachykinin PPT-A gene is not unique in its use of differential exon expression as a mechanism of tissue-specific gene regulation. The gene encoding calcitonin (CT) and calcitonin gene-related peptide (CGRP), as shown from studies of both the rat and human gene, also employs this strategy (Amara et al., 1982; Rosenfeld et al., 1983; Amara et al., 1984; Jonas et al., 1985). The gene is composed of 6 exons (Fig. 1B). Exons 1 - 3 are constitutive and contain the 5’ untranslated and common coding regions. Exon 4 contains the CT coding sequence and a poly(A) addition site and is used in thyroid C cells. In neural tissue exon 3 is spliced instead to exons 5 and 6, which contain the CGRP coding and 3 ’ untranslated sequences and poly(A) addition site. CT functions as a circulating calcium homeostatic hormone (Fontaine et al., 1987) while CGRP appears to have more diverse actions having both neuromodulatory and trophic activities (Rosenfeld, 1983). In addition to coding for CT or CGRP both messages encode two other putative peptides, the functions of which have yet to be elucidated.
A
Neuron-specific alternative RNA splicing Both the calcitonin/CGRP gene and the PPT-A gene employ mechanisms of alternative RNA processing to differentially express mRNAs in a tissuespecific manner, i.e. the alternative mRNAs are expressed in either neural or non-neural tissue. However, the R15 neuropeptide precursor of the marine slug Aplysia generates two different mRNAs by a mechanism of alternative splicing which results in their differential expression within a single tissue, the nervous system (Buck et al., 1987). The two different mRNAs differ in a single region whereby a 6-nucleotide sequence present in one mRNA (R15-1) is replaced by a different 48-
Fig. 2. In situ hybridization showing expression of FMRFamide exon (A) and GDPFLRFamide exon (B) specific sequences in two alternate sections from the Lymnaea brain. In most instances the same cell is visible in both sections. The DNA hybridization probes and procedures are as described in Linacre et al., (1990) and Saunders et al., (1991). ( x 120.)
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an RNA transcript within the Aplysia nervous system can result in differential mRNA expression within individual neurons (Buck et al., 1987). In the freshwater snail Lyrnnaea, the FMRFamide (Phe-Met-Arg-Phe-NH2) related peptides are encoded by two distinct exons (Linacre et al., 1990; Saunders et al., 1991). The question arises as to where these sequences are expressed in the brain. It is possible that both are expressed in the same cells or alternatively each exon could be expressed in a different cell, i.e. expression is exclusive. By the use of in situ hybridization
nucleotide sequence in the alternative mRNA (R15-2). The 48-nucleotide sequence was shown by genomic mapping to be encoded within a single exon and encodes a portion of the a 1 peptide, a peptide isolated from neuron R15 possessing potent osmoregulatory activity. Thus the 48 for 6 nucleotide substitution is likely to be of physiological significance. Many neurons within the abdominal ganglion express the R15 gene, as shown by in situ hybridization, but expression of the 48-nucleotide exon appears to be specific to the R15 neuron (Buck et al., 1987). Thus alternative processing of
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6 x SDPFLRFa 1 x SDPYLRFa 1 x SDPFFRFa 1 x EFFPLa Fig. 3. Model for expression of the FMRFamide gene in Lymnaea. Expression of the FMRFamide-related peptide gene appears to be regulated by alternative splicing of a primary transcript by the "mutually exclusive exon" mode of splicing. In this case the expression is regulated in a neuron-specific manner. The gene consists of two peptide encoding exons encoding the tetrapeptides and heptapeptides. Also shown is a hydrophobic leader sequence (HLl) encoded by another exon which is spliced onto both peptide-encoding exons. It is not clear whether the second hydrophobic leader sequence (R), in-frame with the FMRFamide exon, is expressed. The mature peptides and the number of copies of each peptide sequence are shown. The data for this model is derived from Linacre et al., (1990), Saunders et al., (1991) and Saunders et al. (in press).
pepbdeS
121
with either FMRFamide (tetrapeptide) or GDPFLRFamide (heptapeptide) specific DNA probes, the expression pattern in individual neurons can be determined. Fig. 2 shows alternate sections through the CNS of Lymnaea hybridized to FMRFamide specific sequences (Linacre et al., 1990) or GDPFLRFamide-specific exon sequences (Saunders et al., 1991). It can be seen that cells expressing the FMRFamide exon in the cytoplasm (as judged by the “halo” pattern of hybridization) do not express the GDPFLRFamide exon in the cytoplasm, and vice versa. The FMRFamide-gene has been shown to consist of at least 3 exons (Linacre et al., 1990; Saunders et a]., 1991; Saunders et al., 1992) and appears to produce at least 2 different mature transcripts by a “mutually exclusive” mode of alternative splicing where either the tetrapeptide or heptapeptide - exon (Fig. 3) is spliced into a specific mRNA, exclusion or inclusion of both simultaneously does not occur (Fig. 3; Saunders et al., 1992). However, until the gene has been fully characterized at both the 5’ and 3’ termini the utilization of other mechanisms of alternative RNA processing by this gene, such as differential polyadenylation site usage, cannot be entirely ruled out. In comparison to the tachykinin and calcitonin genes, the spliced transcripts derived from the Lymnaea FMRFamide-related peptide gene do not appear to be differentially expressed in a tissue-specific manner but in a neuron-specific manner; like the expression of the Aplysia R15 precursor encoding gene which has been shown to be regulated in a neuron-specific manner (Buck et al., 1987). Individual neurons within the Lymnaea nervous system also regulate the processing of an RNA transcript from a single gene in different ways (Fig. 2 and Saunders et al., 1992). However, the expression of the FMRFamide-related peptide gene is the clearest example of neuron-specific gene expression shown to date and occurs in a more diverse range of behaviourally related neurons.
Possible mechanisms of regulation of neuronspecific alternative RNA splicing Evidence to date strongly suggests that the Lymnaea FMRFamide-related peptide gene, as is being discovered for an increasing number of genes, employs the strategy of alternative RNA processing to generate diversity from its transcription unit (Fig. 3 and Saunders et al. in press). However, the mechanisms involved in the regulation of alternative processing pathways are presently little understood. Signals must reside in information encoded within the gene transcript (cis-elements), but may require additional control by diffusible (transacting) factors. Cases of alternative splicing where different mRNAs derived from structurally homogeneous primary transcripts are expressed in a cell type- or developmental stage-specific manner, clearly demonstrate that the splicing “environment” of different cells varies for these transcripts. The differences in “environment” are likely to be determined by variability in type or concentration of trans-acting factors. The nature of such factors is for most cases unclear, however all the components of the spliceosome including the snRNAs and other components of the snRNPs are potential candidates. The action of trans-acting factors is likely to affect the folding of the premRNA to present particular donor or acceptor pairs in proximity, or inhibit or promote the usage of a particular splicing junction. In vitro studies have suggested that the secondary structure adopted by RNA molecules can influence splicesite selection, and thus determine the outcome of the splicing event. For example, sequences in the introns at either side of exon 6 in the rat tachykinin PPT-A gene have been proposed to promote intrastrand annealing, forming a hairpin structure which could prevent inclusion of this exon, thus resulting in the formation of a-PPTmRNA. Similarly, sequences adjacent to exon 4 could potentially interact to exclude this exon, thus producing y-
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PPTmRNA (Carter and Krause, 1990). Such a hypothesis is based on the finding that splice sites sequestered in genetically engineered RNA hairpin loops can be omitted in the splicing process (Solnick, 1985; Eperon et al., 1986). However, although such observations established the potential of secondary structure in influencing splicing choices, they did not establish whether such a mechanism is normally used in vivo. It has subsequently been shown that when such constructs were introduced into HeLa cells the hairpin loops had little or no affect on the splicing choices (Solnick and Lee, 1987). These results suggest that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all (possibly due to the actions of endogenous RNA helicases) (Solnick and Lee, 1987). The elements involved in regulating the tissuespecific expression of the rat calcitonin gene have been the focus of several studies. Interestingly, it has been shown that the tissue-specific processing is the result of alternative splice-site selection primarily regulated by cis-active sequences at the calcitonin-specific 3 ’ splice junction, and not controlled by differential polyadenylation site usage (Emerson et al., 1989). The results obtained from this study are consistent with a model in which cisactive sequences serve to inhibit the formation of calcitonin mRNA in CGRP-producing cells, most probably by binding trans-acting factors that are specific to CGRP-producing cells. The nature of such a putative factor(s) has yet to be elucidated. Analysis of the mechanisms operating to regulate the differential processing of the FMRFamiderelated peptide gene transcript will obviously await the complete characterization of the gene. However, from the results shown here (and Saunders et al., 1992) it would appear that different neurons within the CNS offer a different splicing “environment” to the primary transcript. It is possible that one of the spliced transcripts is produced by a “default mode” of splicing and the other by a “regulated mode”, requiring the expression of specific trans-acting factors within those cells ex-
pressing that transcript, but it is also equally possible that both types of splicing reaction require specific factors. Comparison with other FMRFamide related genes The structure of the Aplysia gene has yet to be fully determined, mainly due to the lack of genomic clones. However, analysis of cDNA clones has suggested that the prohormone precursor is encoded within at least two exons (Taussig and Scheller, 1986). A more detailed analysis of the Drosophila FMRFamide-related peptide gene has revealed that this gene consists of two exons separated by a -2.5 kb intervening sequence (Chin et al., 1990; Schneider and Taghert, 1990). However the prohormone precursor is entirely encoded within the second exon, the first exon encodes an untranslated leader sequence. In comparison, the Lymnaea gene consists of at least 3 exons. The first exon encodes a spliced hydrophobic leader sequence which, although translated, does not appear in the prohormone structures. Furthermore, 2 prohormones can derive from this gene, one encoding the tetrapeptides and the other encoding the heptapeptides, both of which are encoded within discrete exons (Saunders et al., submitted). The splicing strategies used by the genes also appear to differ. For example, sequencing of cDNAs derived from the Aplysia gene suggests that the primary transcript can be spliced to produce mature transcripts which vary in relation to the copy number of FMRFamide encoding sequences contained within them (Taussig and Scheller, 1986). The precise nature of the multiple transcripts which have been detected in Aplysia is unclear but it has been proposed that they are the products of “intra-exonic” splicing events. However, it is also possible that the cDNAs are not true representations of the mRNA transcript(s) but are cloning artifacts which arose as the result of recombination between the highly repeated FMRFamide encoding sequences. In favour of the alternative splicing hypothesis is the detection of multiple transcripts that have different sizes within neural tissue (Schaefer et al.,
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1985). However, analysis of the expression of these putative spliced transcripts has not extended to in situ hybridization analysis and thus differential expression of this gene by alternative splicing has not convincingly been shown. Analysis of cDNAs derived from the Lymnaea gene coupled with the in situ hybridization data presented here suggests that the primary precursor derived from this gene is subject to alternative RNA splicing. However, the splicing mechanism used in this case is “interexonic” rather than “intra-exonic” as is possibly the case in Aplysia and thus results in the production of mature transcripts that differ in the nature of peptides encoded rather than the copy number of one peptide as in Aplysia. In contrast t o the molluscan FMRFamide-related peptide genes described, the Drosophila gene appears to give rise to a single transcript and thus the potential for alternative splicing appears slight (Schneider and Taghert, 1988; Schneider and Taghert, 1990; Chin et al., 1990). In addition t o the organization of the genes, the organization and FMRFamide-related peptide content of the prohormones derived from each gene differs. The Aplysia gene encodes 28 copies of FMRFamide, and a single copy of FLRFamide. The Lymnaea gene also encodes these two peptides but the copy number is different: 9 copies of FMRFamide and 2 copies of FLRFamide are encoded within one exon (Linacre et al., 1990). The other exon encodes the heptapeptides GDPFLRFamide and SDPFLRFamide (Saunders et al., 1991). The Drosophila gene, however, does not encode these forms of tetrapeptides and heptapeptides but encodes a diverse range of related peptides, i.e. 13 hepta- and nonapeptides that represent N-terminal extensions of the FMRFamide sequence, the copy number of each individual peptide sequence varies from 1 - 5. Thus although transcription of the Drosophila gene appears to produce less complex transcripts than either the Aplysia or Lymnaea genes, it encodes a more complex repertoire of FMRFamiderelated peptides. The expression of the Drosophila gene has been analysed by both immunocytochemistry (White et al., 1986; Chin et al., 1990) and in situ hybridiza-
tion (Chin et al., 1990), and has been shown to be expressed in - 500 cells within the nervous system. The possibility that the prohormone undergoes differential processing has been suggested from the study by Chin et al., (1990), whereby an antibody generated against a peptide spanning a potential proteolytic cleavage site within the precursor is shown to stain a unique set of cells. However, the specificity of this antibody to the FMRFamide precursor has yet to be conclusively shown and thus the question of whether differential processing actually occurs remains unanswered. Thus, while the splicing strategy of the Drosophila gene appears simple it may use mechanisms of post-translational processing to generate diversity from its transcriptional unit. Whether the prohormones derived from the Lymnaea gene are also subject to differential processing has yet to be determined. One significant similarity between the expression of the FMRFamide-related peptide encoding genes of Lymnaea and Drosphila is their widespread distribution throughout the nervous system, which suggests important physiological roles for FMRFamide-related peptides in both systems. The axonal projections of the 40 - 50 Drosophila FMRFamide-immunoreactive central neurons has suggested that they are involved in a variety of physiological processes (Chin et al., 1990) and include neurosecretory cells which project to release sites in the periphery and interneurons whose processes are confined to the CNS (Schneider and Taghert, 1990). Our data show that the Lymnaea gene is similarly expressed in a wide variety of cell types. However, unlike the Drosophila CNS which because of its small size has not been extensively studied in terms of neuropeptide physiology, the Lymnaea CNS has proved to be an ideal model system in which to perform studies of peptidergic and non-peptidergic neural systems.
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Note added in Pro03 FMRFamide sequences can be found in GENBANK: M87479.
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