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Alternative splicing in the testes
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Alternative splicing in the testes Commentary Julian P Venables Germ-cell differentiation is an ideal process for studying the effects of alternative splicing and there are examples of alternative splicing of genes involved in gene regulation and signal transduction at every stage of the spermatogenic pathway. A network of testes-specific splicing factor interactions has been uncovered and combining our knowledge of these RNAs and proteins should lead to an understanding of the regulation of alternative splicing and male fertility. Address Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; e-mail: [email protected] Current Opinion in Genetics & Development 2002, 12:615–619 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations CREB cAMP response element binding protein CREM cAMP response element modulator protein EST expressed sequence tag GFP green fluorescent protein PK protein kinase PTB polypyrimidine tract binding protein SAF-B scaffold attachment factor B SCF stem cell factor
Introduction Alternative splicing is a mechanism for controlling gene expression in all cell types [1], including testes. In a recent bioinformatics survey of the entire EST database [2••], alternative splicing was found in 40% of all genes and three quarters fell into the broad category of gene regulation and cell-signalling molecules, implying that alternative splicing occurred more by ‘design’ than by accident and that it is an important mechanism of gene regulation. In this Commentary, I set out to find testes-specific alternative splices that are differentially expressed, either in testes as a whole, or during specific stages of spermatogenesis, that could be under the control of a putative testes-specific splicing factor complex [3]. With this aim in mind, only examples of alternative splicing of internal exons (flanked by two splice sites) have been included, as according to ‘exon definition’ alternative external exons must depend on alternative promoters or polyadenlyation sites. Examples have also been omitted if they are not conserved in man or if the mouse orthologue has been knocked out without any apparent affect on fertility. Alternative splicing has hitherto been mostly studied in the brain, which is an extremely complicated organ upon which the whole organism depends [4•,5•]. Mice lacking genes involved in spermatogenesis, however, are usually viable and offer a unique opportunity to study alternative splicing in an entire differentiation process
on one microscope slide [6]. Alternative splicing during spermatogenesis can even now be studied in vitro [7••].
Alternative splicing and sex determination Alternative splicing is critical for sex determination in fruit flies [1], and recent information suggests this may also be the case in mammals. Several proteins including SRY and WT1 are known to act as determinants of phenotypic sex in the course of embryonic development [8]. SRY had been thought to be involved only in regulating transcription, but recent evidence [9••] has demonstrated that it contributes to splicing and is part of the spliceosome. WT1 is another protein that is known to act as a transcription factor. However, alternative splicing of the WT1 gene results in the incorporation of three amino acids (K, T and S) that are thought to convert WT1 from a transcription factor to a splicing factor [10], and this +KTS form is essential for male sex determination in mice [11••]. Early male germ cell development also requires TIAR, an apoptosis-promoting protein that is known to act at (U)-rich intronic enhancers [12].
Alternative splicing of meiotic genes Meiosis is a form of cell division unique to germ cells and two critical meiotic transcripts are alternatively spliced in the testes. DMC1 and MSH4 are proteins associated with the synaptonemal complex, from yeast to humans, and mice without either gene have a block to the zygotene stage of meiosis [13]. Dmc1 pre-mRNA is spliced in male and female germ cells of all mammals to give two isoforms, one with an extra region coding for 55 amino acids, which is lacking in somatic cells. The shorter form is postulated to have lower ATPase activity [14,15]. Msh4 is also alternatively spliced into two forms, one of which has an extra coding region for 58 amino acids [16].
Alternative splicing of gene regulators in the testes There are many testes-specific genes [17] under the control of testes-specific promoters [6], as well as ubiquitous genes that are under various forms of post-transcriptional control [18], including sequestration and delayed translation [19]. Transcripts from several genes that regulate gene expression are themselves alternatively spliced. A testes-specific splice of the Sry-related transcription factor Sox17, which lacks the exon containing the start site, replaces the normal message during male meiosis, and consequently, an inactive N-terminal truncation lacking the DNA-binding domain replaces full-length Sox17 in spermatids [20]. The cAMP-responsive transcription factors CREM and CREB are intricately involved in the continuing process of spermatogenesis [21], and there are at least 23 different
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isoforms resulting from multiple promoters, alternative polyadenlyation and among these, multiple alternative splicing events as well [22]. During male meiosis, there is a switch from active to inactive CREB (by incorporation of a stop codon exon) and from inactive to active CREM (by incorporation of transactivating domains) directed by alternative splicing. However, it remains to be demonstrated that these isoforms are not redundant with the various other activator and inhibitor forms, and that the switch to the activator form of CREB does not just cancel out the switch to the repressor form of CREM.
domains [29]. This alternative splicing must be strongly regulated, as there is no overlap in the expression of the germ cell and somatic forms. The protein kinase C family is involved in cell growth, differentiation and apoptosis. Normally PKCδ is cleaved by caspase 3 between its catalytic and regulatory domains, resulting in deregulation of its catalytic domain. An alternative downstream 5′ splice site is used in testes, which incorporates an extra 78 bases. This results in the addition of 26 amino acids that blocks cleavage by caspase 3. This isoform can also be induced by the apoptosis-inducing chemical H-7 [30].
HSF2 is a transcriptional regulator of the essential spermatogenesis gene hsp70.2. HSF2α has an extra short exon of 54 bases encoding an additional 18 amino acids next to its leucine zipper domain that increases its transactivation function. This exon is not found in HSF2β, which at the protein level is upregulated during early Pachynema [23].
Hormonal aspects of cell signalling are also subject to alternative splicing in the testes. Cytochrome p450 aromatase converts androgens to oestrogen. In rats, it has been reported that there is a shift during spermatogenesis, between the pachytene and round spermatid stages, from a normal 5′ splice site to an upstream alternative splice site, the use of which removes 49 bases causing a frame shift and a truncated and inactive protein [31]. Neprilysin 2 is a hormone metalloprotease expressed only in testes and brain. The testes exclusively express a form containing an extra 37 base exon with a stop codon that encodes a truncated protein lacking the active site [32].
The cold-shock DNA-binding protein, also known as mouse Y-box protein 3, is a dual function transcription factor/translation repressor, which has alternating basic and acidic motifs of unknown function. A testes-specific form has an extra 69-residue base/acid repeat encoded by an alternative 207 base exon [24]. Another alternative splice predicted to affect translation in mouse testes is that of cysteinyl tRNA synthetase. This incorporates a testesspecific exon of 249 bases encoding an 83 amino acid region capable of binding translation initiation factor eIFγ [25]. Alternative splicing can also regulate the control of gene expression at the protein level. The ATE1 protein is involved in selecting proteins for degradation via the ubiquitin pathway by adding arginine to their N termini. The coding region of ATE-1 has two mutually exclusive exons of 129 bases to create ATE1-1p and ATE1-2p. The ratio of these two forms varies considerably between tissues and in mouse testes 90% exists as ATE1-1p whereas in muscle 90% exists as ATE1-2p, and in vitro and in vivo assays suggest that the muscle form may be inactive and excluded from the nucleus [26].
Signal transduction and alternative splicing
Signals that control the mitotic and meiotic cell cycles in spermatocytes have as their effectors molecules involved in nuclear architecture including envelope breakdown and reassembly, and alternative splicing in the testes may be involved in this. AKAP149 is a protein, containing a C-terminal (KH type) RNA-binding domain, that anchors PKA to the nuclear envelope before formation of the nuclear lamina [33]. An alternative form of AKAP149, S-AKAP84 is specifically made in male germ cells by inclusion of an extra exon with a stop codon that removes the KH domain [34]. AKAP84 has been shown recently to bind tubulin and associate with metaphase spindles. It has been postulated that one of the functions of AKAP84 is to enhance phosphorylation by PKA of another tubulin-binding protein, MAP2 [35]. The MAP2 message has three exons, including one that is nearly 4kb in length, spliced (in frame) out of the coding region in rat testes [36] and this internally deleted form has lower affinity for phosphatidylinositol [37]. The nuclear lamina associated peptide LAP2 has two isoforms, β and γ, that are both expressed in round spermatids. Lap2β has an extra 327 base exon encoding an additional 109 amino acids. During the elongation of haploid spermatids, splicing switches to produce exclusively this longer form, and concomitantly the localisation of LAP2 shifts to a point at the centriolar pole of the spermatid nucleus [38].
PAC1R is a seven-transmembrane domain G proteincoupled receptor that stimulates the production of phosphatidylinositol by phospholipase C. A testes-specific variant of PAC1R contains an extra 72 base exon encoding an additional 24 amino acids in the extracellular ligandbinding domain which down-regulates phosphatidylinositol production [27•]. A testes-specific splice of phospholipase C δ4 incorporates an extra 96 nucleotides encoding an additional 32 amino acids, rich in phosphorylation sites, between its catalytic domains in a region postulated to be important for interaction with various signalling components [28].
Somatic splices
C3G is a ubiquitous guanine nucleotide-releasing protein that binds to adaptor protein SH3 domains. The testesspecific variant C3G-2 contains an exon of 153 bases encoding an extra 51 amino acids next to the SH3-binding
Differentiating germ cells are associated with and dependent on the somatic cells in the seminiferous tubules, known as Sertoli cells. Whereas nine out of ten of the examples of simple alternative cassette exons in germ cells discussed above involve exon inclusion, in somatic cells there are five
Alternative splicing in the testes Venables
known examples of alternative splicing, all of which involve exon skipping of important coding regions. The rat E-box-binding transcription factor misses an exon in Sertoli cells and has a 24 amino acid deletion in its protein, which results in higher homodimerization and DNA-binding activity [39]. Prolactin receptor is essential for proper spermatogenesis in the mouse and has been found in an alternatively spliced form in red deer testes, lacking two exons, leading to a frame shift and premature termination. This produces a soluble rather than membrane-bound protein incapable of responding to prolactin [40]. There are also two examples of inhibition of pre-protein cleavage by exon skipping in Sertoli cells. Exogenous stem cell factor (SCF) is necessary and sufficient for initiating meiosis in male germ cells [7••]. A developmental switch to an alternative splice lacking the proteolysis site causes the membrane bound rather than the soluble form of the protein to be made by Sertoli cells, and mouse mutants at the SCF locus that can only make the soluble form of SCF have impaired spermatogenesis [41]. Also in a conserved mammalian splice in Sertoli cells the calpain cleavage site of the pre-protein of interleukin α is omitted so the mature form is not produced. This leads to a stimulation of testosterone production from the other main somatic cell type in the testes, the Leydig cells [42]. The scavenger high-density lipoprotein receptor is expressed in the cell membrane of Leydig cells as well as in other tissues. The C-terminus and stop codon of the type 1 variant are very precisely removed in the type 2 variant resulting in translation of an alternative cytoplasmic domain and this form predominates in the testes [43].
Splice alternators in the testes As well as the many alternative splices that are specific to the various testicular cell types detailed above, there are also some known germ cell-specific proteins that are likely to play a role in alternative splice site selection. HnRNP G and RBMY are homologous RNA-binding proteins from the X and Y chromosomes that are conserved in all mammals and marsupials [44]. In mammals, hnRNP G has undergone retro-transposition and is now expressed from at least two autosomes as well [45]. Two testes-specific forms exist: RBM, which is often deleted in infertile men [46] and hnRNP G-T from chromosome 11, which is mainly expressed during meiosis [47]. A clue that these proteins might have a specific function in alternative splicing is that they interact and co-localise with alternative splicing factor TRA2β and several other ‘RS’ domain-containing proteins, which are crucially important for splicing [48–50]. A testesspecific splice of tra2, caused by TRA2 binding specifically to its own message, is essential for spermatogenesis in Drosophila [51] and there is a similar alternative splice of tra2β in rat testes, but its expression is less restricted [52]. SRPK1, which phosphorylates ‘RS’ domains, is predominantly expressed in the testes and may affect splice site choice by altering the subcellular localisation of splicing
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factors, and hence their effective concentrations [53]. Phosphorylation of TRA2β by SRPK1 reduces non-specific RNA binding and increases its interaction with RBM [48]. RBM also interacts with the ‘signal transduction and RNA processing’ family member T-STAR, which is predominantly expressed in testes [54] and its rat homologue alters splicing of various minigenes by acting at purine-rich sequences [55••]. Significantly SRPK1, hnRNPA1, hnRNP G, T-STAR, and a subset of ‘RS’ domain proteins including SRp30c and ASF/SF2 all interact independently with nuclear scaffold attachment factor B that may therefore nucleate a set of clustered splicing modulators to recruit genes for alternative splicing in selected tissues such as testes and brain [55••,56••,57•].
Conclusions and future prospects There is a lengthening list of alternatively spliced transcripts in the literature that could be formed by testesspecific splicing factors. These are probably the tip of the iceberg, however, as they have mostly been discovered by randomly chosen RT-PCR primers designed to describe the expression of genes as a whole rather than specific isoforms. Many more candidates will be uncovered experimentally, such as by studying interactions of testesspecific splicing factors or by knocking them out, both in vivo and in the newly established in vitro spermatogenesis system [7••]. Differential RNAs can then be used to screen microarrays of oligonucleotides corresponding to exon:exon junctions [58•]. Furthermore, as EST coverage saturates it will become increasingly possible to search for tissuespecific alternative splices in the curated alternative splicing databases [59•]. The commonest kind of alternative splicing is the omission or addition of a discrete (cassette) exon and there is a clear split between somatic cells and germ cells, which are subject to exon skipping and inclusion, respectively. This observation is consistent with the hypothesis that a network of interactions surrounding SAF-B constitutes a germ-cell-specific alternative spliceactivating complex.
Acknowledgements J Venables is supported by a grant from the Wellcome Trust. Thanks to Rachel Davies, Ian Eperon, Howard Cooke and especially David Elliott for comments and discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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Modrek B, Resch A, Grasso C, Lee C: Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001, 29:2850-2859. Modrek et al. have mapped >2 million ESTs to the human genome and found 6200 examples of alternative splicing which they predict to double over the next few years. A random sample of the alternative splices were categorised according to function as follows: membrane proteins 29%, secreted proteins 14%, transcription factors 14%, apoptotic 11% and cell signalling molecules 9%. 3.
Elliott DJ: Splicing and the single cell. Histol Histopathol 2000, 15:239-249.
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4. Grabowski PJ, Black DL: Alternative RNA splicing in the nervous • system. Prog Neurobiol 2001, 65:289-308. Alternative splicing in the brain affects diverse processes such as receptor and channel activation and synaptic localisation. There are cases of changes of alternative splicing in response to neural excitation and involvement of alternative splicing in diseases such as spinal muscular atrophy. Also, as in testes, there are examples of tissue-specific splicing modulators including Nova1, neural PTB, KSRP and SRPK2. (See also [5•]).
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19. Kleene KC: A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells. Mech Dev 2001, 106:3-23.
Dredge BK, Polydorides AD, Darnell RB: The splice of life: alternative splicing and neurological disease. Nat Rev Neurosci 2001, 2:43-50. See annotation [4•]. Venables JP, Cooke HJ: Lessons from knockout and transgenic mice for infertility in men. J Endocrinol Invest 2000, 23:584-591.
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Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, Goldberg E, Dym M: Generation and in vitro differentiation of a spermatogonial cell line. Science 2002, 297:392-395. Feng et al. took spermatogonial cells from 6-day-old mice and immortalised them with telomerase. Then by addition of a single chemical (SCF) to the culture medium, they caused the cell line to initiate spermatogenesis and undergo meiosis on a similar time scale to mice. After 1 week, synaptonemal complexes and crossing over and meiosis-specific marker lactate dehydrogenase C4 were observed. After 2 weeks, protamine 2 was expressed and after 3 weeks more than half the cells were haploid and resembled spermatids. The spermatogonial cell line is readily transfectable and GFP fused to the expression elements of Acrosin was visualised in a tight spot at the cell pole resembling an acrosome. This cell line could be used to analyse the effect of knocking out testes-specific splicing factors or to make transgenic mice by ICSI. 8.
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Ohe K, Lalli E, Sassone-Corsi P: A direct role of SRY and SOX proteins in pre-mRNA splicing. Proc Natl Acad Sci USA 2002, 99:1146-1151. The testes-determining factor Sry is one of ~20 SRY-like (SOX) proteins. SRY co-localises with splicing factors in characteristic nuclear speckles. Ohe et al. prove a functional link between SOX proteins and splicing by blocking splicing in living cells with U6 antisense RNA. This causes an enlargement of the speckles, which still contain SOX6 and splicing factors. In vitro splicing extracts were then depleted with anti-SOX6 antibodies which inhibited splicing of model substrates at the first step and this could be reversed by the addition of recombinant GST-SOX proteins including SRY. The authors speculate that there may be a novel mechanism of specification of the male germline in the XY gonadal ridge, namely alternative splicing mediated by the peculiar DNA-binding domain of SRY that may also bind RNA. 10. Davies RC, Calvio C, Bratt E, Larsson SH, Lamond AI, Hastie ND: WT1 interacts with the splicing factor U2AF65 in an isoformdependent manner and can be incorporated into spliceosomes. Genes Dev 1998, 12:3217-3225. 11. Hammes A, Guo JK, Lutsch G, Leheste JR, Landrock D, Ziegler U, •• Gubler MC, Schedl A: Two splice variants of the Wilms’ tumor 1 gene have distinct functions during sex determination and nephron formation. Cell 2001, 106:319-329. Frasier syndrome is a human genetic disease characterised by, among other things, male to female sex reversal and some Frasier patients have a mutation that leads to reduced use of a downstream alternative 5′ splice site that incorporates three amino acids K, T and S in the WT1 protein. Hammes et al. mimicked this WT1 mutation in mice by changing GT to GC at the splice site so the ‘Frasier’ mice could only make the –KTS isoform and they did indeed observe XY sex reversal in the homozygotes. 12. Le Guiner C, Lejeune F, Galiana D, Kister L, Breathnach R, Stevenin J, Del Gatto-Konczak F: TIA-1 and TIAR activate splicing of alternative exons with weak 5′′ splice sites followed by a U-rich stretch on their own pre-mRNAs. J Biol Chem 2001, 276:40638-40646. 13. Wolgemuth DJ, Laurion E, Lele KM: Regulation of the mitotic and meiotic cell cycles in the male germ line. Recent Prog Horm Res 2002, 57:75-101. 14. Habu T, Taki T, West A, Nishimune Y, Morita T: The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic Acids Res 1996, 24:470-477. 15. Sato S, Seki N, Hotta Y, Tabata S: Expression profiles of a human gene identified as a structural homologue of meiosis-specific recA-like genes. DNA Res 1995, 2:183-186.
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Daniel PB, Kieffer TJ, Leech CA, Habener JF: Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenesis. J Biol Chem 2001, 276:12938-12944. Daniel et al. provide a perfect example of how alternative splicing can have profound implications in the testes. They created stable cell lines expressing two splice forms of PAC1R, a seven transmembrane domain receptor protein that responds to short polypeptide hormones called PACAPs. The testes-specific isoform has an extra exon (3a) just 161 bases downstream of constitutive exon 3, which translates in the extracellular ligand-binding domain. Binding studies of radiolabelled PACAP38 to cells expressing the two PAC1Rs showed a six-fold greater affinity of the 3a-containing receptor. This correlates with a decreased coupling to phospholipase C-dependent signalling pathways as measured by accumulation of inositol. cAMP pathways were however increased in the 3a-containing cell line. Alternative splicing in the testes can therefore cause a switch from inositol to cAMPdependent signalling pathways. 28. Lee SB, Rhee SG: Molecular cloning, splice variants, expression, and purification of phospholipase C-delta 4. J Biol Chem 1996, 271:25-31. 29. Shivakrupa, Singh R, Swarup G: Identification of a novel splice variant of C3G which shows tissue-specific expression. DNA Cell Biol 1999, 18:701-708. 30. Sakurai Y, Onishi Y, Tanimoto Y, Kizaki H: Novel protein kinase C delta isoform insensitive to caspase-3. Biol Pharm Bull 2001, 24:973-977. 31. Levallet J, Mittre H, Delarue B, Carreau S: Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells. J Mol Endocrinol 1998, 20:305-312. 32. Ouimet T, Facchinetti P, Rose C, Bonhomme MC, Gros C, Schwartz JC: Neprilysin II: a putative novel metalloprotease and its isoforms in CNS and testis. Biochem Biophys Res Commun 2000, 271:565-570.
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48. Venables JP, Elliott DJ, Makarova OV, Makarov EM, Cooke HJ, Eperon IC: RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2beta and affect splicing. Hum Mol Genet 2000, 9:685-694. 49. Elliott DJ, Bourgeois CF, Klink A, Stevenin J, Cooke HJ: A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection. Proc Natl Acad Sci USA 2000, 97:5717-5722. 50. Elliott DJ, Oghene K, Makarov G, Makarova O, Hargreave TB, Chandley AC, Eperon IC, Cooke HJ: Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development. J Cell Sci 1998, 111:1255-1265. 51. Mattox W, McGuffin ME, Baker BS: A negative feedback mechanism revealed by functional analysis of the alternative
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isoforms of the Drosophila splicing regulator transformer-2. Genetics 1996, 143:303-314. 52. Nayler O, Cap C, Stamm S: Human transformer-2-beta gene (SFRS10): complete nucleotide sequence, chromosomal localization, and generation of a tissue-specific isoform. Genomics 1998, 53:191-202. 53. Papoutsopoulou S, Nikolakaki E, Chalepakis G, Kruft V, Chevaillier P, Giannakouros T: SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1. Nucleic Acids Res 1999, 27:2972-2980. 54. Venables JP, Vernet C, Chew SL, Elliott DJ, Cowmeadow RB, Wu J, Cooke HJ, Artzt K, Eperon IC: T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis. Hum Mol Genet 1999, 8:959-969. 55. Stoss O, Olbrich M, Hartmann AM, Konig H, Memmott J, Andreadis A, •• Stamm S: The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites. J Biol Chem 2001, 276:8665-8673. SAM68 and the related testicular protein T-STAR, also known as rSLM2, are RNA-binding proteins that bind to cell-signalling molecules such as Ins(1,4,5)P3 kinase and are thought to be important for cell-cycle progression, but previously the mechanism of this action was not known. Stoss et al. showed an interaction of rSLM2 with spliceosome-associated proteins and scaffold attachment factor B (SAF-B), in the yeast two-hybrid system, in in vitro pull-downs and by immunoprecipitation. rSLM2 also caused incorporation of an alternative exon containing a purine-rich enhancer and SLM2 bound specifically to this element. This is the first direct evidence that ‘signal transduction and RNA processing’ (STAR) proteins can act by regulating splicing. 56. Denegri M, Chiodi I, Corioni M, Cobianchi F, Riva S, Biamonti G: •• Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors. Mol Biol Cell 2001, 12:3502-3514. Denegri et al. studied the nuclear localisation of scaffold attachment factor B (aka HAP) and found it in a punctate pattern and these spots were concentrated into larger brighter foci after heat shock or cadmium stress in HeLa cells. This localisation pattern was also observed in a subset of ‘RS’ domain proteins and with SAM68. An ‘ER’-rich region of SAF-B was found both to interact with all these proteins and to be responsible for the nuclear localisation when fused alone to GFP. Stress treatments also caused use of the downstream 5′ splice site of the adenovirus E1A model alternative splicing substrate. The SAF-B bodies might be the sites where alternative splicing occurs in a subset of tissues including testes. 57. •
Nikolakaki E, Kohen R, Hartmann AM, Stamm S, Georgatsou E, Giannakouros T: Cloning and characterization of an alternatively spliced form of SR protein kinase 1 that interacts specifically with scaffold attachment factor-B. J Biol Chem 2001, 276:40175-40182. Nikolaki et al. found a novel alternative splice of the ‘RS’ domain-kinase SRPK1 in testes that had a retained intron of 513 bases between codons 4 and 5 which add an extra 171 amino acids into the N terminus. SRPK1a was more active than ordinary SRPK1 so it may be important despite being expressed at a low level, and significantly an SRPK1a-specific antibody could immmunoprecipitate an ‘RS’-kinase activity from rat testes cytosolic extracts. A yeast two-hybrid screen found that the 171 amino acids interact with scaffold attachment factor B. SRPK1 did not phosphorylate SAF-B, however, so the implication is that SAF-B would tether the kinase in a substrate-binding position. 58. Clark TA, Sugnet CW, Ares M Jr.: Genomewide analysis of mRNA • processing in yeast using splicing-specific microarrays. Science 2002, 296:907-910. Clarke et al. designed oligonucleotides ‘probes’ against exon, intron and exon:exon junctions of the several hundred spliced yeast genes, and printed them onto micro arrays. A global analysis of the effect of splicing of 18 splicing protein yeast mutants was displayed. Although this kind of microarray would have to be enlarged one thousand fold for human exons to be definitively screened, it is likely that many key alternative splices could be simultaneously studied in human tissues by this method in the not so distant future. 59. Modrek B, Lee C: A genomic view of alternative splicing. Nat Genet • 2002, 30:13-19. Modrek and Lee give the ‘big picture’ view of alternative splicing in increasing complexity of the proteome and predict that it will become a major research area to rival transcriptional regulation. This is a ‘one stop’ review for people looking for links to the various alternative splicing databases or who are interested in the appliance of microarrays and bioinformatics to the problems of alternative splicing.
Alternative splicing in the testes Commentary Julian P Venables Germ-cell differentiation is an ideal process for studying the effects of alternative splicing and there are examples of alternative splicing of genes involved in gene regulation and signal transduction at every stage of the spermatogenic pathway. A network of testes-specific splicing factor interactions has been uncovered and combining our knowledge of these RNAs and proteins should lead to an understanding of the regulation of alternative splicing and male fertility. Address Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; e-mail: [email protected] Current Opinion in Genetics & Development 2002, 12:615–619 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations CREB cAMP response element binding protein CREM cAMP response element modulator protein EST expressed sequence tag GFP green fluorescent protein PK protein kinase PTB polypyrimidine tract binding protein SAF-B scaffold attachment factor B SCF stem cell factor
Introduction Alternative splicing is a mechanism for controlling gene expression in all cell types [1], including testes. In a recent bioinformatics survey of the entire EST database [2••], alternative splicing was found in 40% of all genes and three quarters fell into the broad category of gene regulation and cell-signalling molecules, implying that alternative splicing occurred more by ‘design’ than by accident and that it is an important mechanism of gene regulation. In this Commentary, I set out to find testes-specific alternative splices that are differentially expressed, either in testes as a whole, or during specific stages of spermatogenesis, that could be under the control of a putative testes-specific splicing factor complex [3]. With this aim in mind, only examples of alternative splicing of internal exons (flanked by two splice sites) have been included, as according to ‘exon definition’ alternative external exons must depend on alternative promoters or polyadenlyation sites. Examples have also been omitted if they are not conserved in man or if the mouse orthologue has been knocked out without any apparent affect on fertility. Alternative splicing has hitherto been mostly studied in the brain, which is an extremely complicated organ upon which the whole organism depends [4•,5•]. Mice lacking genes involved in spermatogenesis, however, are usually viable and offer a unique opportunity to study alternative splicing in an entire differentiation process
on one microscope slide [6]. Alternative splicing during spermatogenesis can even now be studied in vitro [7••].
Alternative splicing and sex determination Alternative splicing is critical for sex determination in fruit flies [1], and recent information suggests this may also be the case in mammals. Several proteins including SRY and WT1 are known to act as determinants of phenotypic sex in the course of embryonic development [8]. SRY had been thought to be involved only in regulating transcription, but recent evidence [9••] has demonstrated that it contributes to splicing and is part of the spliceosome. WT1 is another protein that is known to act as a transcription factor. However, alternative splicing of the WT1 gene results in the incorporation of three amino acids (K, T and S) that are thought to convert WT1 from a transcription factor to a splicing factor [10], and this +KTS form is essential for male sex determination in mice [11••]. Early male germ cell development also requires TIAR, an apoptosis-promoting protein that is known to act at (U)-rich intronic enhancers [12].
Alternative splicing of meiotic genes Meiosis is a form of cell division unique to germ cells and two critical meiotic transcripts are alternatively spliced in the testes. DMC1 and MSH4 are proteins associated with the synaptonemal complex, from yeast to humans, and mice without either gene have a block to the zygotene stage of meiosis [13]. Dmc1 pre-mRNA is spliced in male and female germ cells of all mammals to give two isoforms, one with an extra region coding for 55 amino acids, which is lacking in somatic cells. The shorter form is postulated to have lower ATPase activity [14,15]. Msh4 is also alternatively spliced into two forms, one of which has an extra coding region for 58 amino acids [16].
Alternative splicing of gene regulators in the testes There are many testes-specific genes [17] under the control of testes-specific promoters [6], as well as ubiquitous genes that are under various forms of post-transcriptional control [18], including sequestration and delayed translation [19]. Transcripts from several genes that regulate gene expression are themselves alternatively spliced. A testes-specific splice of the Sry-related transcription factor Sox17, which lacks the exon containing the start site, replaces the normal message during male meiosis, and consequently, an inactive N-terminal truncation lacking the DNA-binding domain replaces full-length Sox17 in spermatids [20]. The cAMP-responsive transcription factors CREM and CREB are intricately involved in the continuing process of spermatogenesis [21], and there are at least 23 different
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isoforms resulting from multiple promoters, alternative polyadenlyation and among these, multiple alternative splicing events as well [22]. During male meiosis, there is a switch from active to inactive CREB (by incorporation of a stop codon exon) and from inactive to active CREM (by incorporation of transactivating domains) directed by alternative splicing. However, it remains to be demonstrated that these isoforms are not redundant with the various other activator and inhibitor forms, and that the switch to the activator form of CREB does not just cancel out the switch to the repressor form of CREM.
domains [29]. This alternative splicing must be strongly regulated, as there is no overlap in the expression of the germ cell and somatic forms. The protein kinase C family is involved in cell growth, differentiation and apoptosis. Normally PKCδ is cleaved by caspase 3 between its catalytic and regulatory domains, resulting in deregulation of its catalytic domain. An alternative downstream 5′ splice site is used in testes, which incorporates an extra 78 bases. This results in the addition of 26 amino acids that blocks cleavage by caspase 3. This isoform can also be induced by the apoptosis-inducing chemical H-7 [30].
HSF2 is a transcriptional regulator of the essential spermatogenesis gene hsp70.2. HSF2α has an extra short exon of 54 bases encoding an additional 18 amino acids next to its leucine zipper domain that increases its transactivation function. This exon is not found in HSF2β, which at the protein level is upregulated during early Pachynema [23].
Hormonal aspects of cell signalling are also subject to alternative splicing in the testes. Cytochrome p450 aromatase converts androgens to oestrogen. In rats, it has been reported that there is a shift during spermatogenesis, between the pachytene and round spermatid stages, from a normal 5′ splice site to an upstream alternative splice site, the use of which removes 49 bases causing a frame shift and a truncated and inactive protein [31]. Neprilysin 2 is a hormone metalloprotease expressed only in testes and brain. The testes exclusively express a form containing an extra 37 base exon with a stop codon that encodes a truncated protein lacking the active site [32].
The cold-shock DNA-binding protein, also known as mouse Y-box protein 3, is a dual function transcription factor/translation repressor, which has alternating basic and acidic motifs of unknown function. A testes-specific form has an extra 69-residue base/acid repeat encoded by an alternative 207 base exon [24]. Another alternative splice predicted to affect translation in mouse testes is that of cysteinyl tRNA synthetase. This incorporates a testesspecific exon of 249 bases encoding an 83 amino acid region capable of binding translation initiation factor eIFγ [25]. Alternative splicing can also regulate the control of gene expression at the protein level. The ATE1 protein is involved in selecting proteins for degradation via the ubiquitin pathway by adding arginine to their N termini. The coding region of ATE-1 has two mutually exclusive exons of 129 bases to create ATE1-1p and ATE1-2p. The ratio of these two forms varies considerably between tissues and in mouse testes 90% exists as ATE1-1p whereas in muscle 90% exists as ATE1-2p, and in vitro and in vivo assays suggest that the muscle form may be inactive and excluded from the nucleus [26].
Signal transduction and alternative splicing
Signals that control the mitotic and meiotic cell cycles in spermatocytes have as their effectors molecules involved in nuclear architecture including envelope breakdown and reassembly, and alternative splicing in the testes may be involved in this. AKAP149 is a protein, containing a C-terminal (KH type) RNA-binding domain, that anchors PKA to the nuclear envelope before formation of the nuclear lamina [33]. An alternative form of AKAP149, S-AKAP84 is specifically made in male germ cells by inclusion of an extra exon with a stop codon that removes the KH domain [34]. AKAP84 has been shown recently to bind tubulin and associate with metaphase spindles. It has been postulated that one of the functions of AKAP84 is to enhance phosphorylation by PKA of another tubulin-binding protein, MAP2 [35]. The MAP2 message has three exons, including one that is nearly 4kb in length, spliced (in frame) out of the coding region in rat testes [36] and this internally deleted form has lower affinity for phosphatidylinositol [37]. The nuclear lamina associated peptide LAP2 has two isoforms, β and γ, that are both expressed in round spermatids. Lap2β has an extra 327 base exon encoding an additional 109 amino acids. During the elongation of haploid spermatids, splicing switches to produce exclusively this longer form, and concomitantly the localisation of LAP2 shifts to a point at the centriolar pole of the spermatid nucleus [38].
PAC1R is a seven-transmembrane domain G proteincoupled receptor that stimulates the production of phosphatidylinositol by phospholipase C. A testes-specific variant of PAC1R contains an extra 72 base exon encoding an additional 24 amino acids in the extracellular ligandbinding domain which down-regulates phosphatidylinositol production [27•]. A testes-specific splice of phospholipase C δ4 incorporates an extra 96 nucleotides encoding an additional 32 amino acids, rich in phosphorylation sites, between its catalytic domains in a region postulated to be important for interaction with various signalling components [28].
Somatic splices
C3G is a ubiquitous guanine nucleotide-releasing protein that binds to adaptor protein SH3 domains. The testesspecific variant C3G-2 contains an exon of 153 bases encoding an extra 51 amino acids next to the SH3-binding
Differentiating germ cells are associated with and dependent on the somatic cells in the seminiferous tubules, known as Sertoli cells. Whereas nine out of ten of the examples of simple alternative cassette exons in germ cells discussed above involve exon inclusion, in somatic cells there are five
Alternative splicing in the testes Venables
known examples of alternative splicing, all of which involve exon skipping of important coding regions. The rat E-box-binding transcription factor misses an exon in Sertoli cells and has a 24 amino acid deletion in its protein, which results in higher homodimerization and DNA-binding activity [39]. Prolactin receptor is essential for proper spermatogenesis in the mouse and has been found in an alternatively spliced form in red deer testes, lacking two exons, leading to a frame shift and premature termination. This produces a soluble rather than membrane-bound protein incapable of responding to prolactin [40]. There are also two examples of inhibition of pre-protein cleavage by exon skipping in Sertoli cells. Exogenous stem cell factor (SCF) is necessary and sufficient for initiating meiosis in male germ cells [7••]. A developmental switch to an alternative splice lacking the proteolysis site causes the membrane bound rather than the soluble form of the protein to be made by Sertoli cells, and mouse mutants at the SCF locus that can only make the soluble form of SCF have impaired spermatogenesis [41]. Also in a conserved mammalian splice in Sertoli cells the calpain cleavage site of the pre-protein of interleukin α is omitted so the mature form is not produced. This leads to a stimulation of testosterone production from the other main somatic cell type in the testes, the Leydig cells [42]. The scavenger high-density lipoprotein receptor is expressed in the cell membrane of Leydig cells as well as in other tissues. The C-terminus and stop codon of the type 1 variant are very precisely removed in the type 2 variant resulting in translation of an alternative cytoplasmic domain and this form predominates in the testes [43].
Splice alternators in the testes As well as the many alternative splices that are specific to the various testicular cell types detailed above, there are also some known germ cell-specific proteins that are likely to play a role in alternative splice site selection. HnRNP G and RBMY are homologous RNA-binding proteins from the X and Y chromosomes that are conserved in all mammals and marsupials [44]. In mammals, hnRNP G has undergone retro-transposition and is now expressed from at least two autosomes as well [45]. Two testes-specific forms exist: RBM, which is often deleted in infertile men [46] and hnRNP G-T from chromosome 11, which is mainly expressed during meiosis [47]. A clue that these proteins might have a specific function in alternative splicing is that they interact and co-localise with alternative splicing factor TRA2β and several other ‘RS’ domain-containing proteins, which are crucially important for splicing [48–50]. A testesspecific splice of tra2, caused by TRA2 binding specifically to its own message, is essential for spermatogenesis in Drosophila [51] and there is a similar alternative splice of tra2β in rat testes, but its expression is less restricted [52]. SRPK1, which phosphorylates ‘RS’ domains, is predominantly expressed in the testes and may affect splice site choice by altering the subcellular localisation of splicing
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factors, and hence their effective concentrations [53]. Phosphorylation of TRA2β by SRPK1 reduces non-specific RNA binding and increases its interaction with RBM [48]. RBM also interacts with the ‘signal transduction and RNA processing’ family member T-STAR, which is predominantly expressed in testes [54] and its rat homologue alters splicing of various minigenes by acting at purine-rich sequences [55••]. Significantly SRPK1, hnRNPA1, hnRNP G, T-STAR, and a subset of ‘RS’ domain proteins including SRp30c and ASF/SF2 all interact independently with nuclear scaffold attachment factor B that may therefore nucleate a set of clustered splicing modulators to recruit genes for alternative splicing in selected tissues such as testes and brain [55••,56••,57•].
Conclusions and future prospects There is a lengthening list of alternatively spliced transcripts in the literature that could be formed by testesspecific splicing factors. These are probably the tip of the iceberg, however, as they have mostly been discovered by randomly chosen RT-PCR primers designed to describe the expression of genes as a whole rather than specific isoforms. Many more candidates will be uncovered experimentally, such as by studying interactions of testesspecific splicing factors or by knocking them out, both in vivo and in the newly established in vitro spermatogenesis system [7••]. Differential RNAs can then be used to screen microarrays of oligonucleotides corresponding to exon:exon junctions [58•]. Furthermore, as EST coverage saturates it will become increasingly possible to search for tissuespecific alternative splices in the curated alternative splicing databases [59•]. The commonest kind of alternative splicing is the omission or addition of a discrete (cassette) exon and there is a clear split between somatic cells and germ cells, which are subject to exon skipping and inclusion, respectively. This observation is consistent with the hypothesis that a network of interactions surrounding SAF-B constitutes a germ-cell-specific alternative spliceactivating complex.
Acknowledgements J Venables is supported by a grant from the Wellcome Trust. Thanks to Rachel Davies, Ian Eperon, Howard Cooke and especially David Elliott for comments and discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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2. ••
Modrek B, Resch A, Grasso C, Lee C: Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001, 29:2850-2859. Modrek et al. have mapped >2 million ESTs to the human genome and found 6200 examples of alternative splicing which they predict to double over the next few years. A random sample of the alternative splices were categorised according to function as follows: membrane proteins 29%, secreted proteins 14%, transcription factors 14%, apoptotic 11% and cell signalling molecules 9%. 3.
Elliott DJ: Splicing and the single cell. Histol Histopathol 2000, 15:239-249.
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4. Grabowski PJ, Black DL: Alternative RNA splicing in the nervous • system. Prog Neurobiol 2001, 65:289-308. Alternative splicing in the brain affects diverse processes such as receptor and channel activation and synaptic localisation. There are cases of changes of alternative splicing in response to neural excitation and involvement of alternative splicing in diseases such as spinal muscular atrophy. Also, as in testes, there are examples of tissue-specific splicing modulators including Nova1, neural PTB, KSRP and SRPK2. (See also [5•]).
16. Santucci-Darmanin S, Paul R, Michiels JF, Saunieres A, Desnuelle C, Paquis-Flucklinger V: Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues. Mamm Genome 1999, 10:423-427. 17.
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19. Kleene KC: A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells. Mech Dev 2001, 106:3-23.
Dredge BK, Polydorides AD, Darnell RB: The splice of life: alternative splicing and neurological disease. Nat Rev Neurosci 2001, 2:43-50. See annotation [4•]. Venables JP, Cooke HJ: Lessons from knockout and transgenic mice for infertility in men. J Endocrinol Invest 2000, 23:584-591.
7. ••
Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, Goldberg E, Dym M: Generation and in vitro differentiation of a spermatogonial cell line. Science 2002, 297:392-395. Feng et al. took spermatogonial cells from 6-day-old mice and immortalised them with telomerase. Then by addition of a single chemical (SCF) to the culture medium, they caused the cell line to initiate spermatogenesis and undergo meiosis on a similar time scale to mice. After 1 week, synaptonemal complexes and crossing over and meiosis-specific marker lactate dehydrogenase C4 were observed. After 2 weeks, protamine 2 was expressed and after 3 weeks more than half the cells were haploid and resembled spermatids. The spermatogonial cell line is readily transfectable and GFP fused to the expression elements of Acrosin was visualised in a tight spot at the cell pole resembling an acrosome. This cell line could be used to analyse the effect of knocking out testes-specific splicing factors or to make transgenic mice by ICSI. 8.
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Ohe K, Lalli E, Sassone-Corsi P: A direct role of SRY and SOX proteins in pre-mRNA splicing. Proc Natl Acad Sci USA 2002, 99:1146-1151. The testes-determining factor Sry is one of ~20 SRY-like (SOX) proteins. SRY co-localises with splicing factors in characteristic nuclear speckles. Ohe et al. prove a functional link between SOX proteins and splicing by blocking splicing in living cells with U6 antisense RNA. This causes an enlargement of the speckles, which still contain SOX6 and splicing factors. In vitro splicing extracts were then depleted with anti-SOX6 antibodies which inhibited splicing of model substrates at the first step and this could be reversed by the addition of recombinant GST-SOX proteins including SRY. The authors speculate that there may be a novel mechanism of specification of the male germline in the XY gonadal ridge, namely alternative splicing mediated by the peculiar DNA-binding domain of SRY that may also bind RNA. 10. Davies RC, Calvio C, Bratt E, Larsson SH, Lamond AI, Hastie ND: WT1 interacts with the splicing factor U2AF65 in an isoformdependent manner and can be incorporated into spliceosomes. Genes Dev 1998, 12:3217-3225. 11. Hammes A, Guo JK, Lutsch G, Leheste JR, Landrock D, Ziegler U, •• Gubler MC, Schedl A: Two splice variants of the Wilms’ tumor 1 gene have distinct functions during sex determination and nephron formation. Cell 2001, 106:319-329. Frasier syndrome is a human genetic disease characterised by, among other things, male to female sex reversal and some Frasier patients have a mutation that leads to reduced use of a downstream alternative 5′ splice site that incorporates three amino acids K, T and S in the WT1 protein. Hammes et al. mimicked this WT1 mutation in mice by changing GT to GC at the splice site so the ‘Frasier’ mice could only make the –KTS isoform and they did indeed observe XY sex reversal in the homozygotes. 12. Le Guiner C, Lejeune F, Galiana D, Kister L, Breathnach R, Stevenin J, Del Gatto-Konczak F: TIA-1 and TIAR activate splicing of alternative exons with weak 5′′ splice sites followed by a U-rich stretch on their own pre-mRNAs. J Biol Chem 2001, 276:40638-40646. 13. Wolgemuth DJ, Laurion E, Lele KM: Regulation of the mitotic and meiotic cell cycles in the male germ line. Recent Prog Horm Res 2002, 57:75-101. 14. Habu T, Taki T, West A, Nishimune Y, Morita T: The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic Acids Res 1996, 24:470-477. 15. Sato S, Seki N, Hotta Y, Tabata S: Expression profiles of a human gene identified as a structural homologue of meiosis-specific recA-like genes. DNA Res 1995, 2:183-186.
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Daniel PB, Kieffer TJ, Leech CA, Habener JF: Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenesis. J Biol Chem 2001, 276:12938-12944. Daniel et al. provide a perfect example of how alternative splicing can have profound implications in the testes. They created stable cell lines expressing two splice forms of PAC1R, a seven transmembrane domain receptor protein that responds to short polypeptide hormones called PACAPs. The testes-specific isoform has an extra exon (3a) just 161 bases downstream of constitutive exon 3, which translates in the extracellular ligand-binding domain. Binding studies of radiolabelled PACAP38 to cells expressing the two PAC1Rs showed a six-fold greater affinity of the 3a-containing receptor. This correlates with a decreased coupling to phospholipase C-dependent signalling pathways as measured by accumulation of inositol. cAMP pathways were however increased in the 3a-containing cell line. Alternative splicing in the testes can therefore cause a switch from inositol to cAMPdependent signalling pathways. 28. Lee SB, Rhee SG: Molecular cloning, splice variants, expression, and purification of phospholipase C-delta 4. J Biol Chem 1996, 271:25-31. 29. Shivakrupa, Singh R, Swarup G: Identification of a novel splice variant of C3G which shows tissue-specific expression. DNA Cell Biol 1999, 18:701-708. 30. Sakurai Y, Onishi Y, Tanimoto Y, Kizaki H: Novel protein kinase C delta isoform insensitive to caspase-3. Biol Pharm Bull 2001, 24:973-977. 31. Levallet J, Mittre H, Delarue B, Carreau S: Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells. J Mol Endocrinol 1998, 20:305-312. 32. Ouimet T, Facchinetti P, Rose C, Bonhomme MC, Gros C, Schwartz JC: Neprilysin II: a putative novel metalloprotease and its isoforms in CNS and testis. Biochem Biophys Res Commun 2000, 271:565-570.
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38. Alsheimer M, Fecher E, Benavente R: Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins. J Cell Sci 1998, 111:2227-2234. 39. Chaudhary J, Kim G, Skinner MK: Expression of the basic helix-loop-helix protein REBalpha in rat testicular Sertoli cells. Biol Reprod 1999, 60:1244-1250. 40. Jabbour HN, Clarke LA, Bramley T, Postel-Vinay MC, Kelly PA, Edery M: Alternative splicing of the prolactin receptor gene generates a 1.7 kb RNA transcript that is linked to prolactin function in the red deer testis. J Mol Endocrinol 1998, 21:51-59. 41. Mauduit C, Chatelain G, Magre S, Brun G, Benahmed M, Michel D: Regulation by pH of the alternative splicing of the stem cell factor pre-mRNA in the testis. J Biol Chem 1999, 274:770-775. 42. Sultana T, Svechnikov K, Weber G, Soder O: Molecular cloning and expression of a functionally different alternative splice variant of prointerleukin-1alpha from the rat testis. Endocrinology 2000, 141:4413-4418. 43. Webb NR, de Villiers WJ, Connell PM, de Beer FC, van der Westhuyzen DR: Alternative forms of the scavenger receptor BI (SR-BI). J Lipid Res 1997, 38:1490-1495. 44. Elliott DJ: RBMY genes and AZFb deletions. J Endocrinol Invest 2000, 23:652-658. 45. Lingenfelter PA, Delbridge ML, Thomas S, Hoekstra HE, Mitchell MJ, Graves JA, Disteche CM: Expression and conservation of processed copies of the RBMX gene. Mamm Genome 2001, 12:538-545. 46. Elliott DJ, Millar MR, Oghene K, Ross A, Kiesewetter F, Pryor J, McIntyre M, Hargreave TB, Saunders PT, Vogt PH et al.: Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm. Proc Natl Acad Sci USA 1997, 94:3848-3853. 47.
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48. Venables JP, Elliott DJ, Makarova OV, Makarov EM, Cooke HJ, Eperon IC: RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2beta and affect splicing. Hum Mol Genet 2000, 9:685-694. 49. Elliott DJ, Bourgeois CF, Klink A, Stevenin J, Cooke HJ: A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection. Proc Natl Acad Sci USA 2000, 97:5717-5722. 50. Elliott DJ, Oghene K, Makarov G, Makarova O, Hargreave TB, Chandley AC, Eperon IC, Cooke HJ: Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development. J Cell Sci 1998, 111:1255-1265. 51. Mattox W, McGuffin ME, Baker BS: A negative feedback mechanism revealed by functional analysis of the alternative
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isoforms of the Drosophila splicing regulator transformer-2. Genetics 1996, 143:303-314. 52. Nayler O, Cap C, Stamm S: Human transformer-2-beta gene (SFRS10): complete nucleotide sequence, chromosomal localization, and generation of a tissue-specific isoform. Genomics 1998, 53:191-202. 53. Papoutsopoulou S, Nikolakaki E, Chalepakis G, Kruft V, Chevaillier P, Giannakouros T: SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1. Nucleic Acids Res 1999, 27:2972-2980. 54. Venables JP, Vernet C, Chew SL, Elliott DJ, Cowmeadow RB, Wu J, Cooke HJ, Artzt K, Eperon IC: T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis. Hum Mol Genet 1999, 8:959-969. 55. Stoss O, Olbrich M, Hartmann AM, Konig H, Memmott J, Andreadis A, •• Stamm S: The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites. J Biol Chem 2001, 276:8665-8673. SAM68 and the related testicular protein T-STAR, also known as rSLM2, are RNA-binding proteins that bind to cell-signalling molecules such as Ins(1,4,5)P3 kinase and are thought to be important for cell-cycle progression, but previously the mechanism of this action was not known. Stoss et al. showed an interaction of rSLM2 with spliceosome-associated proteins and scaffold attachment factor B (SAF-B), in the yeast two-hybrid system, in in vitro pull-downs and by immunoprecipitation. rSLM2 also caused incorporation of an alternative exon containing a purine-rich enhancer and SLM2 bound specifically to this element. This is the first direct evidence that ‘signal transduction and RNA processing’ (STAR) proteins can act by regulating splicing. 56. Denegri M, Chiodi I, Corioni M, Cobianchi F, Riva S, Biamonti G: •• Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors. Mol Biol Cell 2001, 12:3502-3514. Denegri et al. studied the nuclear localisation of scaffold attachment factor B (aka HAP) and found it in a punctate pattern and these spots were concentrated into larger brighter foci after heat shock or cadmium stress in HeLa cells. This localisation pattern was also observed in a subset of ‘RS’ domain proteins and with SAM68. An ‘ER’-rich region of SAF-B was found both to interact with all these proteins and to be responsible for the nuclear localisation when fused alone to GFP. Stress treatments also caused use of the downstream 5′ splice site of the adenovirus E1A model alternative splicing substrate. The SAF-B bodies might be the sites where alternative splicing occurs in a subset of tissues including testes. 57. •
Nikolakaki E, Kohen R, Hartmann AM, Stamm S, Georgatsou E, Giannakouros T: Cloning and characterization of an alternatively spliced form of SR protein kinase 1 that interacts specifically with scaffold attachment factor-B. J Biol Chem 2001, 276:40175-40182. Nikolaki et al. found a novel alternative splice of the ‘RS’ domain-kinase SRPK1 in testes that had a retained intron of 513 bases between codons 4 and 5 which add an extra 171 amino acids into the N terminus. SRPK1a was more active than ordinary SRPK1 so it may be important despite being expressed at a low level, and significantly an SRPK1a-specific antibody could immmunoprecipitate an ‘RS’-kinase activity from rat testes cytosolic extracts. A yeast two-hybrid screen found that the 171 amino acids interact with scaffold attachment factor B. SRPK1 did not phosphorylate SAF-B, however, so the implication is that SAF-B would tether the kinase in a substrate-binding position. 58. Clark TA, Sugnet CW, Ares M Jr.: Genomewide analysis of mRNA • processing in yeast using splicing-specific microarrays. Science 2002, 296:907-910. Clarke et al. designed oligonucleotides ‘probes’ against exon, intron and exon:exon junctions of the several hundred spliced yeast genes, and printed them onto micro arrays. A global analysis of the effect of splicing of 18 splicing protein yeast mutants was displayed. Although this kind of microarray would have to be enlarged one thousand fold for human exons to be definitively screened, it is likely that many key alternative splices could be simultaneously studied in human tissues by this method in the not so distant future. 59. Modrek B, Lee C: A genomic view of alternative splicing. Nat Genet • 2002, 30:13-19. Modrek and Lee give the ‘big picture’ view of alternative splicing in increasing complexity of the proteome and predict that it will become a major research area to rival transcriptional regulation. This is a ‘one stop’ review for people looking for links to the various alternative splicing databases or who are interested in the appliance of microarrays and bioinformatics to the problems of alternative splicing.