The splicing machinery is a genetic modifier of disease severity

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Concluding remarks Our understanding of the molecular mechanisms of Hox protein function has long been focused on the control of its DNA-binding properties by the Exd and Hth cofactors, whose vertebrate counterparts are Pbx and Meis proteins. Although there are some exceptions [24,25], these cofactors are required for a wide range of Hox protein function. The characterization of Hox intrinsic domains and of additional Hox molecular partners now points to the existence of a novel class of cofactors that are only required for a subset of Hox protein activity. The emerging picture (Figure 3) is that these spatially restricted factors, selectively recruited by target-specific cis-regulatory sequences, provide contextual information required for setting up the transregulatory properties of Hox proteins. These contextual Hox cofactors act as intermediate regulatory molecules, adding a layer of regulation between Hox proteins and transcriptional co-activators or co-repressors. This enables fine-tuning of Hox protein activity, eventually leading to the amazing functional diversity Hox proteins achieve in development and evolution. Acknowledgements We are grateful to R.S. Mann for providing us with the picture used in Figure 2a and thank A. Saurin for critical reading of the manuscript.

References 1 Gehring, W.J. et al. (1994) Homeodomain-DNA recognition. Cell 78, 211–223 2 McGinnis, W. and Krumlauf, R. (1992) Homeobox genes and axial patterning. Cell 68, 283–302 3 Gellon, G. and McGinnis, W. (1998) Shaping animal body plans in development and evolution by modulation of Hox expression patterns. BioEssays 20, 116–125 4 Hombria, J.C. and Lovegrove, B. (2003) Beyond homeosis – HOX function in morphogenesis and organogenesis. Differentiation 71, 461–476 5 Graba, Y. et al. (1997) Drosophila Hox complex downstream targets and the function of homeotic genes. BioEssays 19, 379–388 6 Immerglu¨ck, K. et al. (1990) Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62, 261–268 7 Grienenberger, A. et al. (2003) Tgf(beta) signaling acts on a Hox response element to confer specificity and diversity to Hox protein function. Development 130, 5445–5455

8 Rozowski, M. and Akam, M. (2002) Hox gene control of segmentspecific bristle patterns in Drosophila. Genes Dev. 16, 1150–1162 9 Brodu, V. et al. (2002) abdominal A specifies one cell type in Drosophila by regulating one principal target gene. Development 129, 2957–2963 10 Lohmann, I. and McGinnis, W. (2002) Hox genes: it’s all a matter of context. Curr. Biol. 12, R514 11 Gebelein, B. et al. (2004) Direct integration of Hox and segmentation gene inputs during Drosophila development. Nature 431, 653–659 12 Ronshaugen, M. et al. (2002) Hox protein mutation and macroevolution of the insect body plan. Nature 415, 914–917 13 Merabet, S. et al. (2003) The hexapeptide and linker regions of the AbdA Hox protein regulate its activating and repressive functions. Dev. Cell 4, 761–768 14 Gebelein, B. et al. (2002) Specificity of distalless repression and limb primordia development by abdominal hox proteins. Dev. Cell 3, 487–498 15 Mann, R.S. and Affolter, M. (1998) Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423–429 16 Chen, Y. et al. (2004) Direct interaction with Hoxd proteins reverses Gli3-repressor function to promote digit formation downstream of Shh. Development 131, 2339–2347 17 Joulia, L. et al. (2005) Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts. Dev. Biol. 278, 496–510 18 Kobayashi, M. et al. (2001) Groucho augments the repression of multiple Even skipped target genes in establishing parasegment boundaries. Development 128, 1805–1815 19 Andrioli, L.P. et al. (2002) Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms. Development 129, 4931–4940 20 Tolkunova, E.N. et al. (1998) Two distinct types of repression domain in engrailed: one interacts with the groucho corepressor and is preferentially active on integrated target genes. Mol. Cell. Biol. 18, 2804–2814 21 Capovilla, M. and Botas, J. (1998) Functional dominance among Hox genes: repression dominates activation in the regulation of Dpp. Development 125, 4949–4957 22 Piper, D.E. et al. (1999) Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation. Cell 96, 587–597 23 Passner, J.M. et al. (1999) Structure of a DNA-bound ultrabithoraxextradenticle homeodomain complex. Nature 397, 714–719 24 Lohmann, I. et al. (2002) The Drosophila Hox gene deformed sculpts head morphology via direct regulation of the apoptosis activator reaper. Cell 110, 457–466 25 Galant, R. et al. (2002) Hox repression of a target gene: extradenticleindependent, additive action through multiple monomer binding sites. Development 129, 3115–3126 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.07.001

The splicing machinery is a genetic modifier of disease severity Malka Nissim-Rafinia and Batsheva Kerem Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, Israel

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic Corresponding author: Kerem, B. ([email protected]). Available online 20 July 2005 www.sciencedirect.com

modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.

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conductance regulator (CFTR) gene [6]. The 3849C10 kb C/T mutation (a C/T substitution, 10 kb downstream of nucleotide 3849, the last nucleotide of exon 19 in CFTR) generates a cryptic donor splice site in intron 19, which leads to partial inclusion of cryptic 84-bp ‘exon’, containing an in-frame stop codon. Overexpression of the splicing factors Htra2-b1 and SC35 promoted skipping of the 84-bp ‘exon’ and led to a significant increase (w10%) in the level of correctly spliced RNA that was transcribed from the endogenous CFTR gene. Importantly, chloride efflux measurements showed that this increase led to activation of the CFTR channel and restoration of its function [6]. These results provide direct evidence that the level of correctly spliced transcripts, regulated by the splicing machinery, determines the protein function and explains the correlation between the level of correctly spliced CFTR transcripts and disease severity in patients. The role of the splicing machinery as a genetic modifier was studied in two mice strains that are homozygous for the same splicing mutation in the Scn8a gene [7]. Scn8a encodes the sodium channel Nav1.6, which is expressed throughout the nervous system. The splicing mutation is caused by a 4-bp deletion in the donor-splice site of exon three, leading to partial skipping of exons two and three (Figure 1). This results in truncated, nonfunctional Nav1.6 proteins. Although the two strains have the same splicing mutation, they dramatically differ in the disease phenotype. One mouse strain, C3H, has a chronic movement disorder, whereas the other strain, C57BL/6J, has a severe lethal neurological disease. The differences in the phenotype correlated with the Scn8a splicing pattern. In the milder affected C3H strain, 10% of the transcripts are

Introduction A significant proportion (20–30%) of disease-causing mutations in humans affects pre-mRNA splicing (reviewed in Ref. [1]). 10–15% disrupt intronic splicing motifs and an additional 10–15%, located in the coding region (i.e. missense, nonsense or frameshift mutations), disrupt exonic splicing motifs (reviewed in Ref. [2]). Splicing mutations can lead to a mixture of aberrantly and correctly spliced transcripts, by partial skipping of exons or inclusion of intronic sequences, or can change the ratio of programmed alternatively spliced isoforms. Altered patterns of splicing have also been observed in the absence of mutations in the affected genes. The level of correctly spliced transcript or the ratio of splicing isoforms varies among individuals and correlates with disease severity. Hence a role for splicing regulation as a genetic modifier was suggested [3].

Splicing modulation of alleles carrying splicing mutations The overexpression of various splicing factors modulates the splicing pattern of RNA that was transcribed from introduced minigenes carrying splicing mutations, which are present in several inherited diseases, such as cystic fibrosis (CF), spinal muscular atrophy (SMA) and Frontotemporal dementia and parkinson linked to chromosome 17 disorder (FDTP-17) [4–6]. However, whether this modulation can affect the protein function and disease phenotype in cells or animal models carrying splicing defects in disease genes remained unknown. We have recently analyzed a CF-derived nasalpolyp epithelial cell-line carrying the 3849C10 kb C/T splicing mutation in the cystic fibrosis transmembrane Splicing factor R187X ∆SCNM1

Modulated gene

Splicing pattern (%)

gtaaca

∆Scn8a 1

481

2

3

4

95%

5% ∆Scn8a gtaaca ∆Scn8a SCNM1

1

2

3

4

90% 10%

R187X ∆SCNM1

Scn8a gtaagtaaca Normal Scn8a 1

2

3

4

100%

SCNM1 TRENDS in Genetics

Figure 1. Modulation of the splicing pattern of Scn8a by the splicing factor SCNM1. A 4-bp deletion (gtaa) in the donor site of exon three of Scn8a results in the skipping of exons two and three, leading to truncated proteins being expressed. In C3H mice that are homozygous for the normal SCNM1, 10% of the transcripts are correctly spliced. However, in C57BL/6J mice that are homozygous for a stop mutation (R187X) in SCNM1, only 5% are correctly spliced. The exons are shown as blue boxes. Scn8a and SCNM1 are shown in blue and purple, respectively. The thickness of the arrows represents the splicing level (%). www.sciencedirect.com

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correctly spliced, whereas in the severely affected C57BL/6J strain, only 5% are correctly spliced. Positional cloning led to the identification of a modifier gene, SCNM1, a predicted member of the U1C subfamily of RNA-binding proteins that has a role in recognition of donor splice sites. The milder affected strain was found to carry normal alleles of SCNM1, whereas the severely affected mice carried a stop mutation predicted to produce abnormal SCNM1 proteins (Figure 1). Transgenic expression of wild-type SCNM1 in C57BL/6J mice resulted in a 5% increase in the level of correctly spliced Scn8a transcripts, and rescued the lethal phenotype. These results provide evidence that genetic variations which determines the level of functional splicing factors, regulate the splicing of genes carrying splicing mutations and lead to a significant phenotypic variability. Modulation of developmentally programmed alternative splicing Changes in the level of the splicing factors can also regulate the splicing pattern of RNA transcribed from normal alleles of disease-associated genes. This was found in myotonic dystrophy (DM), a multi-systemic disorder caused by two different microsatellite expansions. DM1 is caused by a CUG trinucleotide expansion in the 3 0 untranslated region of the DMPK gene and DM2 is caused by a CCUG expansion in intron one of the ZNF9 gene. The mechanism of the DM pathology was unknown until the discovery of misregulated, alternative splicing of several genes that were suggested to cause DM. Binding assays revealed that several CUG-binding proteins might be involved, including the splicing factors muscleblind-like (MBNL) and CUG-binding protein 1 (CUG-BP1). The expansion of CUG or CCUG-repeats was suggested to lead to sequestration and up-regulation of the MBNL and CUG-BP1 factors, respectively, and hence cause RNA-splicing abnormalities in their natural target genes, among which are the DM-associated genes. Evidence for this hypothesis came from three mouse models presenting with DM features. In one model, the MBNL gene was knocked out, in another CUG-BP1 was overexpressed, and in the third an expanded CUG repeat, inserted into the human a-actin gene, was expressed. In all mice models, foetal splicing isoforms of DM-associated genes were found in neonates or adult mice, indicating a lack of developmental transition from the foetal to the adult splicing pattern [8–10]. Thus, changes in the level of splicing factors can modulate the level of developmentally regulated spliced RNA that is transcribed from the normal alleles of disease-associated genes, resulting in the disease phenotype. Modulation of tissue-specific alternative splicing Changes in splicing regulation were suggested to have a significant role as a genetic modifier in diseases that are caused by changes in the ratio of tissue-specific alternatively spliced isoforms [3]. In vivo support for this hypothesis came from studies in knockout (KO) mice models. A heart-specific KO of the essential SR protein, ASF/SF2, led to alteration in the splicing pattern of three genes in the mutant heart [11]. One of these genes is the calmodulin-dependent kinase IId (CaMKIId), which www.sciencedirect.com

undergoes programmed alternative splicing to generate tissue-specific CaMKIId spliced isoforms. In the ASF/SF2 KO mice, a heart-specific transition from the cardiac to the neuronal spliced CaMKIId isoform resulted in severe cardiomyopathy and early lethality. Overexpression of the neuronal isoform in a transgenic heart also resulted in lethality of the mice, indicating that the phenotype of both mice resulted from changes in tissue-specific alternative splicing of CaMKIId. In another mouse model, the neuronal specific splicing factor, Nova-1, was knocked out. This led to changes in the ratio of tissue-specific spliced isoforms of several genes. The Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons [12,13]. Thus, a decrease in the level of specific splicing factors can modulate the splicing pattern of programmed alternatively spliced genes leading to disease. Interestingly, mutations in splicing factors that affect their activity can regulate the splicing of tissue-specific genes. This was shown in retinitis pigmentosa, in which mutations in the splicing factor PRPF31 inhibit the splicing of the photoreceptor-specific gene, Rhodopsin, resulting in photoreceptor cell-death [14]. Mutations in other splicing factors, such as HPRP3, PRPC8 and SMN, were also found to associate with human diseases (reviewed in Ref. [15]). However, the genes modulated by these factors have yet to be found.

Can abnormal splicing patterns in human disorders be corrected? We summarized the available data revealing association between the level of correctly spliced transcripts and disease severity previously [3]. Together with the studies described here (Table 1) it became apparent that in many genetic diseases rather subtle differences in the splicing pattern can lead to striking differences in protein function and disease phenotype. This might be relevant for the development of therapeutic approaches for many human diseases using different methodologies (reviewed in Ref. [15]). One such approach involves the use of small molecules. We have recently analyzed the effect of sodium butyrate (NaBu) on the CFTR function in CF-derived cells carrying the 3849C10 kb C/T mutation. NaBu is a histone deacetylase inhibitor previously shown to upregulate the expression of splicing factors, such as Htra2-b1 and SR proteins [16,17]. In this study, we showed a NaBu concentration-dependent increase in the level of correctly spliced CFTR RNA. This increase led to activation of the CFTR channel and restoration of its function, similar to the effect obtained by overexpression of splicing factors [6]. Additional small molecules were recently studied in cell-lines derived from familial dysautonomia (FD) and SMA patients carrying splicing mutations. In these studies, aclarubicin, sodium vanadate, valproic acid, (-)-epigallocatechin gallate (EGCG) and kinetin, were shown to modulate the splicing pattern of the endogenous RNA transcribed from the mutant alleles of IKBKAP and SMN genes and to increase the level of IKAP and SMN proteins [17–21].

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Table 1. Splicing factors that affect the level of splicing transcripts leading to diseasea Splicing factor Htra2-b1 SC35 SCNM1

a

MBNL

Disease CF CF Lethal neurological disease DM

CUG-BP1

DM

ASF/SF2

Lethal cardiomyopathy

Nova-1

Lethal motor deficit

Affected gene CFTR CFTR Scn8a

Mutation 3849C10Kb C/T 3849C10Kb C/T Dgtaa

Affected exon C 84-bp ‘exon’ C 84-bp ‘exon’ K Exons 2 and 3

Organism Human Human Mouse

Analyzed tissue Nasal epitheliumb Nasal epitheliumb Brain

Refs [6] [6] [7]

Clcn1 Tnnt3 Tnnt2 Tnnt2 Mtmr1 Clcn1 Mtmr1 CaMKIId

C Exon 7a C Exon F C Exon 5 C Exon 5 K Exon 2.2 C Exon 7a K Exons 2.1 and 2.2 C dA isoform

Mouse

Muscle Muscle Heart Heart

[8,10] [8] [8] [9]

Mouse

Heart

[11]

GlyRa2 GABAA JNK2 Neogenin Gephyrin

K E3A/E3B K g2L/g2S K 6a/6b C Exon 27 C Exon 9

Mouse

Brain

[12]

Mouse

Muscle

[13]

Abbreviations: CF, cystic fibrosis; DM, myotonic dystrophy. A cell-line was analyzed.

b

Concluding remarks Altogether, the results presented in this article provide evidence that the splicing machinery is a genetic modifier of single-gene genetic disorders. The splicing machinery might also have a significant role as a genetic modifier of complex traits, which are highly prevalent in many human populations. Indeed, candidate genes influencing susceptibility to complex diseases such as Asthma, Alzheimer’s and autoimmune diseases were found to produce alternatively spliced isoforms of specific genes, the ratio of which varies between normal and affected individuals [22–24]. Natural genetic variations in the splicing machinery might contribute to the predisposition of different individuals to human diseases and to the severity of their phenotype. The current progress in understanding the role of splicing modulation as a genetic modifier opens new avenues towards developing treatments for many human diseases. Because O70% of the human multi-exon genes are alternatively spliced and produce different functional proteins [25], and proteins that were initially identified to have a role in transcription or translation were found to function as splicing factors [26], it is reasonable to assume that splicing regulation is an important genetic modifier. References 1 Faustino, N.A. and Cooper, T.A. (2003) Pre-mRNA splicing and human disease. Genes Dev. 17, 419–437 2 Cartegni, L. et al. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 3 Nissim-Rafinia, M. and Kerem, B. (2002) Splicing regulation as a potential genetic modifier. Trends Genet. 18, 123–127 4 Hofmann, Y. et al. (2000) Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc. Natl. Acad. Sci. U. S. A. 97, 9618–9623 5 Wang, Y. et al. (2005) Tau exons 2 and 10, which are misregulated in neurodegenerative diseases, are partly regulated by silencers which bind a complex comprised of SRp30c and SRp55 that either recruits or antagonizes htra2beta 1. J Biol Chem 6 Nissim-Rafinia, M. et al. (2004) Restoration of the cystic fibrosis transmembrane conductance regulator function by splicing modulation. EMBO Rep. 5, 1071–1077 7 Buchner, D.A. et al. (2003) SCNM1, a putative RNA splicing factor that modifies disease severity in mice. Science 301, 967–969 www.sciencedirect.com

8 Kanadia, R.N. et al. (2003) A muscleblind knockout model for myotonic dystrophy. Science 302, 1978–1980 9 Ho, T.H. et al. (2005) Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy. Hum. Mol. Genet. 14, 1539–1547 10 Mankodi, A. et al. (2002) Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol. Cell 10, 35–44 11 Xu, X. et al. (2005) ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell 120, 59–72 12 Jensen, K.B. et al. (2000) Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron 25, 359–371 13 Ule, J. et al. (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302, 1212–1215 14 Yuan, L. et al. (2005) Mutations in PRPF31 inhibit pre-mRNA splicing of rhodopsin gene and cause apoptosis of retinal cells. J. Neurosci. 25, 748–757 15 Garcia-Blanco, M.A. et al. (2004) Alternative splicing in disease and therapy. Nat. Biotechnol. 22, 535–546 16 Chang, J.G. et al. (2001) Treatment of spinal muscular atrophy by sodium butyrate. Proc. Natl. Acad. Sci. U. S. A. 98, 9808–9813 17 Brichta, L. et al. (2003) Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. Hum. Mol. Genet. 12, 2481–2489 18 Andreassi, C. et al. (2001) Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum. Mol. Genet. 10, 2841–2849 19 Zhang, M.L. et al. (2001) An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA. Gene Ther. 8, 1532–1538 20 Anderson, S.L. et al. (2003) EGCG corrects aberrant splicing of IKAP mRNA in cells from patients with familial dysautonomia. Biochem. Biophys. Res. Commun. 310, 627–633 21 Slaugenhaupt, S.A. et al. (2004) Rescue of a human mRNA splicing defect by the plant cytokinin kinetin. Hum. Mol. Genet. 13, 429–436 22 Ueda, H. et al. (2003) Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423, 506–511 23 Laitinen, T. et al. (2004) Characterization of a common susceptibility locus for asthma-related traits. Science 304, 300–304 24 Bertram, L. et al. (2005) Family-based association between Alzheimer’s disease and variants in UBQLN1. New Engl. J. Med. 352, 884–894 25 Johnson, J.M. et al. (2003) Genome-wide survey of human alternative premRNA splicing with exon junction microarrays. Science 302, 2141–2144 26 Burckin, T. et al. (2005) Exploring functional relationships between components of the gene expression machinery. Nat. Struct. Mol. Biol. 12, 175–182 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.07.005