The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing

r 2006, Copyright the Authors Journal compilation r 2006, International Society of Differentiation

Differentiation (2006) 74:65–80 DOI:10.1111/j.1432-0436.2006.00060.x

R E V IE W

Maya Pascual . Marta Vicente . Lidon Monferrer . Ruben Artero

The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing

Received October 18, 2005; accepted in revised form December 4, 2005

Abstract Alternative splicing is widely used to generate protein diversity and to control gene expression in many biological processes, including cell fate determination and apoptosis. In this review, we focus on the Muscleblind family of tissue-specific alternative splicing regulators. Muscleblind proteins bind pre-mRNA through an evolutionarily conserved tandem CCCH zinc finger domain. Human Muscleblind homologs MBNL1, MBNL2 and MBNL3 promote inclusion or exclusion of specific exons on different pre-mRNAs by antagonizing the activity of CUG-BP and ETR-3-like factors (CELF proteins) bound to distinct intronic sites. The relative activities of Muscleblind and CELF proteins control a key developmental switch. Defined transcripts follow an embryonic splice pattern when CELF activity predominates, whereas they follow an adult pattern when Muscleblind activity prevails. Human MBNL proteins show functional specializations. While MBNL1 seems to promote muscle differentiation, MBNL3 appears to function in an opposing manner inhibiting expression of muscle differentiation markers. MBNL2, on the other hand, participates in a new RNA-dependent protein localization mechanism involving recruitment of integrin a3 protein to focal adhesions. Both muscleblind mutant Drosophila embryos and Mbnl1 knockout mice show muscle abnormalities and altered splicing of specific transcripts. In addition to regulating terminal muscle differentiation through Maya Pascual
Marta Vicente
Lidon Monferrer
Ruben . ) Artero (* Department of Genetics University of Valencia Doctor Moliner, 50, 46100 Burjasot Valencia Spain Tel: 134 96 3543005 Fax: 134 96 3543029 E-mail: [email protected] U.S. Copyright Clearance Center Code Statement:

alternative splicing control, results by several groups suggest that Muscleblind participates in the differentiation of photoreceptors, neurons, adipocytes and blood cell types. Misregulation of MBNL activity can lead to human pathologies. Through mechanisms not completely identified yet, expression of transcripts containing large non-coding CUG or CCUG repeat expansions mimics muscleblind loss-of-function phenotypes. Archetypical within this class of disorders are myotonic dystrophies. Our understanding of the biology of Muscleblind proteins has increased dramatically over the last few years, but several key issues remain unsolved. Defining the mechanism of the activity of Muscleblind proteins, their splicing partners, and the functional relevance of its several protein isoforms are just a few examples. Key words Muscleblind
myotonic dystrophy
zinc finger proteins
cell differentiation
development

Introduction Development entails the deployment of a cell type-specific set of proteins whose functional properties must be exquisitely orchestrated. Alternative pre-mRNA splicing, the process by which multiple mRNAs can be generated from the same pre-mRNA by the differential joining of 5 0 and 3 0 splice sites, has emerged as a powerful and versatile mechanism to generate a significant fraction of such functionally distinct proteins. As an example, complete functional domains are deleted or added from the protein coding sequence of a large number of apoptotic factors, which generate splice variant products with completely different roles in life and death decisions (Schwerk and Schulze-Osthoff, 2005). It is not surprising, then, that cells evolved developmental

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programs to control alternative splicing of defined sets of transcripts similar to control gene transcription. Regulation of tissue-specific alternative splicing depends primarily on a number of regulatory proteins expressed only in certain tissues. Pan-neural expression of Drosophila ELAV (for embryonic-lethal, abnormal visual system), for example, promotes inclusion of a neural-specific terminal exon in neuroglian pre-mRNAs (Maniatis and Tasic, 2002). Alternative splicing is not only spatially regulated but also temporally coupled with development. The prototypical SR splicing regulator ASF/SF2 has been recently shown to regulate a restricted set of tissue-specific alternative splicing events during programmed remodeling of murine cardiac muscle around 4 weeks after birth to account for increased workload (Xu et al., 2005). Changes in splice site selection, finally, do not require stable differences between specific cell types. They occur normally within a cell in response to numerous stimuli, such as growth factors, hormones and cell depolarization. Although very little is known about how signal transduction pathways impinge on the splicing reaction, three major mechanisms have been described so far: synthesis, phosphorylation and change in localization of splicing regulatory proteins (for recent reviews see Stamm, 2002; Shin and Manley, 2004; Pelisch et al., 2005). Unprogrammed changes in the relative levels of alternative spliced isoforms affect cellular functions. Indeed, a significant proportion (20%–30%) of diseasecausing mutations in humans affects pre-mRNA splicing. While some of these mutations interfere with the function of normal cis-acting splice sequences, others affect the basal splicing machinery or regulators of alternative splicing (Faustino and Cooper, 2003). Overall, splicing defects have been linked to cellular transformation, metastasis and hereditary diseases including retinitis pigmentosa, spinal muscular atrophy and myotonic dystrophy (reviewed in Grabowski and Black, 2001; Cartegni et al., 2002; Faustino and Cooper, 2003; Musunuru, 2003; Nissim-Rafinia and Kerem, 2005). In a broad sense, splicing regulation involves regulatory proteins that interact with specific sequences within pre-mRNAs and subsequently stimulate or repress exon recognition. These proteins bind either directly to 5 0 or 3 0 splice sites or to other pre-mRNA sequences called intronic or exonic splicing enhancers and silencers. Enhancers and silencers stimulate or repress splice-site selection. Despite the fact that the mechanism of alternative pre-mRNA splicing has been the object of intense investigation (Maniatis and Tasic, 2002; Singh, 2002; Black, 2003), relatively few examples of alternative splicing have been fully characterized, and the selection of tissue-specific splicing factors known is clearly lacking. In this review, we summarize the molecular and cell biology of the Muscleblind family of proteins, a newly described family of tissue-specific alternative splicing regulators. We also explore some

other potential molecular functions proposed for these proteins as well as their evolutionary history, and how changes in the activity of Muscleblind proteins help explain the deleterious effect of mutant transcripts bearing non-coding trinucleotide expansions.

Muscleblind proteins are evolutionarily conserved proteins characterized by CCCH tandem zinc finger domains The structurally distinct feature of Muscleblind proteins is the presence of tandem zinc finger domains composed of three cysteine and one histidine residue (herein abbreviated as CCCH; Begemann et al., 1997; Miller et al., 2000; Fardaei et al., 2002; Squillace et al., 2002). The CCCH domain was first recognized in the murine Tristetraprolin protein (TTP; also known as Nup475 and TIS11) by K.D. Brown and coworkers (Gomperts et al., 1990), and since then more than 1600 proteins have been found to contain such domains (Pfam database; http:// www.sanger.ac.uk/Software/Pfam/). Sequence alignment and phylogenetic reconstruction of most of these proteins defined several groups according to the number of zinc fingers present and the spacing of cysteine residues within these domains (Fardaei et al., 2002). These groups included the archetypical mouse TTP, which is involved in the destabilization of defined mRNAs by binding to so-called class II AU-rich elements (ARE) within the 3 0 untranslated region (reviewed in Blackshear, 2002). CCCH domains coordinate zinc atoms (Worthington et al., 1996) and bind single-stranded RNA molecules in a sequence-specific fashion. The NMR structure of the TIS11d tandem zinc finger domain (a member of the TTP family of proteins) bound to the ARE sequence was recently determined (Hudson et al., 2004). Stacking interactions between conserved aromatic side chains in the tandem zinc finger domain and the RNA bases, as well as hydrogen bonds complementary to the Watson–Crick edges of the bases, stabilize the complex. Consistently, mutations in the tandem zinc finger domain of TTP result in a loss of RNA binding and disrupt protein function (Lai et al., 1999; Lai et al., 2002). Although no detailed reconstruction of the Muscleblind protein phylogeny has been attempted yet, the analysis of the published information and the more than 90 protein entries available in public databases (http:// www.ncbi.nih.gov/entrez; May, 2005) leads to some important conclusions. Highly conserved Muscleblind proteins are present in both protostomes, including Nematoda, and deuterostomes (Table 1; Begemann et al., 1997), but not in bacteria, fungi or plants. Thus, Muscleblind proteins are exclusive to Metazoans and are relatively modern, having appeared perhaps 800 million years ago.

67 Table 1 Muscleblind-like sequences from several representative Metazoa species Organism Nematoda Caenorhabditis elegans Arthropoda Anopheles gambiae Apis mellifera Bombyx mori Drosophila melanogaster Tunicata Ciona intestinalis Vertebrata Gallus gallus (chicken)

Genome

Accession number

Protein

Complete

NP_510746.1

Muscleblind

WGS WGS WGS Complete

Reconstructed Reconstructed Reconstructed AAC01949.1

Muscleblind Muscleblind Muscleblind Muscleblind

Complete

Reconstructed

Muscleblind

WGS

CAG31624.1 XP_416979.1 NP_001012591.1 BAA24858 NP_997187.1 AAH74776 NP_064391.2 NP_997398.1 NP_598924.1 XP_342253.2 XP_214253.2 XP_228685.2 SINFRUP00000169845 SINFRUT00000165687 AA584613 SINFRUP00000176586 SINFRUP00000133529 ENSXETP00000046798 ENSXETP00000050703 ENSXETP00000011905

MBNL1 MBNL2 MBNL3 MBNL1 MBNL2 MBNL3 MBNL1 MBNL2 MBNL3 MBNL1 MBNL2 MBNL3 MBNL1 MBNL1a MBNL2 MBNL2a MBNL3 MBNL1 MBNL2 MBNL3

Homo sapiens (human)

Complete

Mus musculus (mouse)

Complete

Rattus norvergicus (rat)

Incomplete

Takifugu rubripes (fish)

WGS

Xenopus tropicalis (frog)

Incomplete

C C C C

Accession numbers are from GenBank except for Takifugu and Xenopus for which the Ensembl accession numbers are given (http:// www.ensembl.org/). When whole-genome annotations were not available, Muscleblind proteins were constructed from sequence similarity searches (Tblastn program) against genomic DNA sequences using the Drosophila Muscleblind sequence as query (indicated as ‘‘reconstructed’’). For arthropods, we specify the Drosophila Muscleblind protein isoform showing homology within the isoform-specific region. For vertebrates, we indicate the human gene showing the highest degree of homology. Drosophila Muscleblind was formerly known as Mindmelt (Kania et al., 1995).

The genomes of all invertebrate species analyzed, from nematodes (Caenorhabditis elegans) to primitive chordates (Ciona intestinalis), contain a single muscleblind gene. Most vertebrate species, however, contain three homologs, with the remarkable exception of spiny fishes (Takifugu rubripes) that contain up to five muscleblind-related sequences (Table 1). Three different genes encode human Muscleblind proteins, which are currently named MBNL1, MBNL2 and MBNL3 (Kanadia et al., 2003b; also known as MBNL/EXP, MBLL/MPL1 and MBXL/CHCR, respectively). Ubiquitous expression of a full-length MBNL1 cDNA in Drosophila muscleblind mutant embryos rescues lethality and a characteristic abdomen hypercontraction phenotype (see below; Monferrer and Artero, 2006). Therefore, human and Drosophila Muscleblind proteins are functionally exchangeable in vivo, thus supporting that they are orthologs i.e., homologs produced by speciation. An examination of the genomic organization of all three human muscleblind genes reveals a very similar exon composition in both number of exons and exon

size (Fardaei et al., 2002). In sharp contrast, the genomic organization of the Drosophila muscleblind gene shows no conservation when compared with its human counterparts. The fact that a primitive Chordate (C. intestinalis) contains a single muscleblind gene, together with the high degree of conservation between the MBNL genes at the sequence and genomic organization level, indicates that human MBNL genes are paralogs. These genes probably arose as a product of a relatively modern gene duplication event from a common ancestor, and subsequently acquired specialized functions. While MBNL1 seems involved in promoting muscle differentiation, MBNL3 appears to function in an opposing manner, and MBNL2 participates in integrin a3 subcellular localization (see below; Miller et al., 2000; Squillace et al., 2002; Adereth et al., 2005). Within the phylogenetic tree, not only does the number of muscleblind genes increase with the biological complexity, but also the domain composition of the proteins encoded. Muscleblind proteins from protostome species contain two CCCH zinc fingers with a typical spacing CX7CX6CX3H between the zinc-binding

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Fig. 1 Sequence comparison among Muscleblind family members. (A) Similarity tree between human and Drosophila Muscleblind zinc fingers. Zinc fingers are named as follows: Hs (Homo sapiens) or Dm (Drosophila melanogaster) followed by the letter N, L or X to indicate MBNL1, MBNL2 or MBNL3, respectively, and a number denoting the position of the specific zinc finger along the protein. The first Drosophila zinc finger (Dm1) clusters with the first and third zinc finger of all MBNL proteins, whereas the second (Dm2) clusters with the second and forth zinc fingers of MBNLs. Dm1 shows higher similarity to the first zinc finger of all MBNL proteins, while Dm2 is more similar to the fourth human zinc finger. (B) Schematic illustration of representative protostome and deuterostome Muscleblind proteins indicating CCCH domain com-

position, typical spacing between zinc-binding moieties and linker lengths between domains. (C) Amino acid alignment within the first two CCCH zinc fingers from flies (Dm), Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), human (Homo sapiens; Hs), Gallus gallus (Gg), Xenopus tropicalis (Xt), Takifugu rubripes (Tr) and Ciona intestinalis (Ci). In vertebrates, only the MBNL1 protein was used in multiple sequence alignments. Within the region shown, C. elegans is the most distantly related Muscleblind protein sharing about 67% identity with MBNL1, whereas vertebrate proteins are almost identical (over 99% identical residues). Besides the CCCH zinc finger domain, Muscleblind proteins show highly conserved sequences, such as the LEV and NGR boxes (boxed in the consensus sequence).

moieties. In contrast, deuterostome genomes (particularly Chordates) encode for proteins with four such domains (Miller et al., 2000; Fardaei et al., 2002; Squillace et al., 2002) in which the second and forth zinc finger are slightly shorter showing a CX7CX4CX3H spacing (Fig. 1B). As we will discuss later, alternative splicing of Drosophila and human muscleblind genes generates protein isoforms containing just one or two zinc fingers, respectively. A comparative analysis of the protein structure in protostomes and deuterostomes is consistent with ancestral proteins having just two CCCH zinc fingers. This pair might have undergone tandem duplication during the speciation of vertebrates to give rise to the modern Muscleblind proteins (Figs. 1A, 1B). Worth noting is the fact that the muscleblind ortholog of a primitive chordate, Ciona intestinalis, contains four CCCH zinc fingers, thus suggesting that the tandem duplication might have

occurred before the divergence between urochordates and vertebrates, approximately 550 million years ago. Alternatively, ancestral Muscleblind proteins might have had four CCCH domains that protostomes reduced to two by deletion of the intervening sequences. A comparison of Muscleblind protein sequences from evolutionary distant species reveals protein motifs other than the CCCH zinc finger (Fig. 1C). The first motif is the consensus sequence WLXLEV, where X indicates any amino acid, and is located immediately before the first zinc finger. We named this sequence a LEV box. The second motif consists of an NGR core sequence immediately followed by either a valine or an asparagine residue, with two additional highly conserved apolar residues before and after the NGR core. We refer to this motif as an NGR box (consensus sequence is V/IX0-2NGRV/NXA/L). Two NGR boxes

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Fig. 2 Schematic representation of the linear structure of several Muscleblind proteins showing salient features. A horizontal line represents Muscleblind proteins from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Ciona intestinalis (Ci) and Homo sapiens (Hs) with features discussed in the text highlighted as shown in the legend. All four Drosophila Muscleblind protein isoforms are represented (MblA-D). Alternative splice protein isoforms MBNL1_v3, MBNL2_v2 and MBNL3_v1 exemplify human MBNL proteins. Characteristic for the whole family is the presence of tandem CCCH zinc fingers and a number of predicted serine/

threonine (CK2 and PKC) phosphorylation sites. In most cases, the putative phosphorylation sites lay within CCCH zinc fingers, thus suggesting a potential regulatory role over RNA-binding capacity. Also conserved in all Muscleblind proteins is a potential WW domain recognition site. Some Muscleblind protein isoforms include low complexity regions such as alanine-rich (CeMbl, DmMblB, HsMBNL1, HsMBNL2), phenylalanine-rich (DmMblB) and proline-rich (HsMBNL2, HsMBNL3) regions of unknown function. Protein length and features are shown to scale.

flank the second zinc finger of all Muscleblind proteins studied. The functional relevance of these motifs, however, is unknown. An additional structural element typical of Muscleblind proteins is the conservation of four aromatic residues within the first zinc finger pair sequence as well as a tryptophan residue within the LEV box (Fig. 1C). CCCH tandem zinc finger domains from TIS11d protein bind ARE sequences by intercalating a tyrosine and a phenylalanine sidechain between the UU and AU dinucleotides, respectively. The hydrophobic stacking of aromatic rings and heterocyclic bases provides the basis of sequence recognition (Hudson et al., 2004; reviewed in Brown, 2005). Because conserved aromatic residues in Muscleblind proteins occupy positions equivalent to the relevant aromatic residues in the TIS11d-ARE complexes, these residues constitute prime candidates to mediate the sequence specificity of Muscleblind binding to RNA targets. Apart from zinc fingers, Muscleblind proteins often contain different low complexity regions, such as alanine-, phenylalanine- and proline-rich regions, which

are conserved to different degrees among the Muscleblind family members (Fig. 2; Squillace et al., 2002; Kino et al., 2004). The functional relevance of such domains is unclear, although proline-rich domains have been extensively associated with protein–protein interactions (Kay et al., 2000). Muscleblind proteins also contain a number of conserved phosphorylation sites, which are potential targets for intracellular signaling pathways. Within CCCH zinc finger domains, Muscleblind proteins show potential phosphorylation target sites for casein kinase 2 (CK2) and protein kinase C (Fig. 2; Monferrer and Artero, 2006). Given their location within the RNA-binding domain, it is plausible that phosphorylation might provide regulation by means of steric hindrance and/or interference by the charged phosphate group. Recently, Mayeda and collaborators provided evidence that CK2 protein kinase activity directly influences pre-mRNA splicing through site-specific phosphorylation of the splicing factor RNPS1 (Trembley et al., 2005). Highly conserved within the proline-rich region is a class IV WW domain interaction motif, which is a phosphoserine and

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phosphothreonine-dependent interaction (Kay et al., 2000). WW domain-containing signal transducers could interact with phosphorylated serine or threonine target sites in Muscleblind proteins to either inhibit or activate its activity as splicing regulators.

Alternative splicing contributes to Muscleblind protein diversity Comprehensive descriptions of alternative splicing from muscleblind genes are available for Drosophila (Begemann et al., 1997; Flybase, http://www.flybase.org) and mammals (Fardaei et al., 2002; Kanadia et al., 2003a; Kino et al., 2004), although the functional adaptations that such structurally diverse Muscleblind protein isoforms provide remain to be addressed experimentally. Primary Drosophila muscleblind transcripts follow a complex alternative splicing pattern that generates four different transcript isoforms, mblA, B, C and D, with common 5 0 ends and specific downstream sequences (Fig. 2). Once conceptually translated, the proteins encoded by mblA, B and C transcripts share the first 179 amino-terminal amino acids, which include two complete CCCH zinc fingers (Begemann et al., 1997; Flybase, http://www.flybase.org). Isoform mblD shares only the first two exons and encodes a small protein 84 amino acids long with one complete zinc finger and conserved LEV and NGR boxes. Because MblD could potentially bind to RNA in an unproductive way, it is tempting to propose that MblD could play a regulatory function. This possibility, however, has no experimental support thus far. Sequence searches within arthropods using isoform-specific sequences as query only detect sequences homologous to MblC thus suggesting that MblC is the most ancestral protein isoform within Arthropoda (Table 1). In addition to the four mblA–D canonical mRNAs, Drosophila muscleblind primary transcripts give rise to RNA molecules with constitutions other than those of typically spliced transcripts. Two such atypical RNA molecules are characterized by containing an incomplete exon 2 tandem repetition (mblE2E2 0 , where the prime symbol indicates an incomplete exon) or having exons scrambled when compared with the corresponding genomic DNA (mblE2E3 0 E2 0 ; Houseley and Artero, unpublished). These transcripts are small (below 1 kb), very abundant, show some developmental regulation, and have been proposed to be circular and non-coding. Such non-canonical transcripts have previously been described only in vertebrates, but their functional implications remain unclear both in vertebrates and in Drosophila. Alternative splicing of primary transcripts from the human MBNL1, MBNL2 and MBNL3 genes gives rise to at least nine, three and six protein isoforms, respectively (Fig. 3; Fardaei et al., 2002; Kino et al., 2004).

Overall, the majority of protein isoforms from MBNL1 and MBNL2 genes contain two complete zinc finger pairs at normal distance from one another. However, exclusion of exon 1 in MBNL1 generates transcripts that encode protein isoforms with a single zinc finger pair, whereas exclusion of exon 3, which encodes the linker region between the first and the second zinc finger pair, juxtaposes both RNA-binding motifs. Isoforms MBNL1_v4 and MBNL1_v5, lacking exon 3, are conserved in mouse, thus suggesting that they play important functions in vivo (Kanadia et al., 2003a). Also remarkable is the small (83 amino acids long) MBNL1_v8 protein isoform that only contains the first zinc finger, LEV and NGR boxes, and an unconserved C-terminus. This spliceform is structurally identical to Drosophila MblD (Fig. 2), and ESTs from cow and mouse confirm that it is evolutionarily conserved. Contrary to MBNL1 and MBNL2, most MBNL3 spliceforms (MBNL3_v3-6) encode for protein isoforms lacking the first two zinc fingers. Deletion studies in a yeast three-hybrid assay and UV crosslinking experiments showed that protein isoforms lacking any zinc finger domain, or the linker region between the zinc finger pairs, do not interact with long CUG trinucleotide repeats (see below) and may similarly be unable to bind target RNAs (Kanadia et al., 2003a; Kino et al., 2004). Worth noting is the fact that the relative abundance of the different splice forms from all three MBNL genes is largely unknown, thus precluding any hypothesis as to which protein isoform may perform the most prevalent role in specific cell types, or if the abundance of different MBNL protein isoforms in the cell of interest has any functional significance. In contrast to the obvious functional implications that the presence or absence of zinc finger motifs may have, the use of other alternative exons in MBNL transcripts seems to cause far weaker effects. Alternative use of exons 5 and 7 of MBNL1, for example, keeps the open reading frame and only changes 18 and 12 amino acids, respectively. Usage of other alternative exons causes frameshifts, but those spliceforms that result in frameshifts are poorly represented in comparison with the inframe spliceforms (Fardaei et al., 2002). In summary, at least 10 MBNL protein isoforms differ in regions other than the zinc finger domains and linker region. For these isoforms, alternative splicing does not control the presence or absence of clearly defined functional domains and their relevance remains to be established.

Muscleblind proteins regulate alternative exon choice during terminal muscle differentiation CCCH domain-containing proteins play pivotal roles in several aspects of RNA metabolism. TTP, for instance, promotes deadenylation and instability of the tumor

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necrosis factor a transcripts (reviewed in Blackshear, 2002; Lai et al., 2002). The C. elegans Pharynx and intestine in excess protein 1 (PIE-1), another CCCH family member, blocks the activity of the positive transcription elongation factor b (P-TEFb) subsequently

inhibiting the progression through the transcriptional elongation step (Zhang et al., 2003). Although Muscleblind proteins were thought to participate in the RNA metabolism from the initial description of the muscleblind gene (Begemann et al., 1997), it was the characterization of an Mbnl1 knockout mouse that first raised the possibility that these proteins played a direct role in splice selection (Kanadia et al., 2003a). Because MBNL proteins were implicated in the neuromuscular disease myotonic dystrophy (see below), the alternative splicing of a number of terminal muscle differentiation transcripts was tested for mis-splicing events. Skeletal muscle ion-transporter chloride channel 1 (Clcn-1), cardiac troponin T (cTNT; not expressed in mature skeletal muscle) and fast skeletal muscle troponin T (Tnnt3) followed aberrant alternative splicing. In most cases, defective alternative splicing consisted in the abnormal retention of ‘‘fetal’’ exons in adult mice (cTNT and TNT3) or the inclusion of cryptic exons (Clcn-1). Direct proof that human MBNL proteins regulate alternative splicing was attained by studying the splicing pattern of exon 5 of the cTNT gene and exon 11 of the insulin receptor (IR) gene (Cooper and Ordahl, 1985; Savkur et al., 2001). In these experiments, all three MBNL proteins similarly regulated splicing of human and chicken cTNT and IR minigenes in MBNL-transfected cell cultures (Ho et al., 2004). MBNL proteins acted as either activators or repressors

Fig. 3 Alternative splicing of human MBNL primary transcripts generates extensive protein diversity. Boxes connected by a horizontal line at the top of each panel represent exons and genomic DNA, respectively, of MBNL1 (A), MBNL2 (B) and MBNL3 (C) genes. Exon–intron structure was taken from the literature and was manually refined and updated by comparing cDNA and genomic sequences (Fardaei et al., 2002; Kino et al., 2004; Pascual and Artero, unpublished). Some exons are not present in all MBNL genes, as indicated. Broken lines connecting specific exon combinations denote the pattern of alternative splicing of human MBNL genes. Only coding exons are represented for alternatively spliced transcripts. In some instances, the use of an alternative exon restores the frameshift generated by the exclusion of an exon upstream. Exclusion of exon 6 in MBNL1_v6, for example, generates a frameshift that is compensated by the inclusion of exon 9. Relevant structural elements, such as the CCCH zinc finger domain and low complexity regions, are indicated as shown in the bottom. There is EST evidence that MBNL1 and MBNL3 transcripts can use alternative 5 0 UTR initiation exons (not represented). Splice variants are named following recommendations by the Human Gene Nomenclature Committee (Wain et al., 2002) and numbered, whenever possible, according to the corresponding Entrez Gene entry (GeneID: MBNL1, 4154; MBNL2, 10150; MBNL3, 55796) with the equivalences that follow. For MBNL1: v1, NM_021038; v2, NM_207292; v3, NM_207293; v4, NM_207294; v5, NM_207295; v6, NM_207296; v7, NM_207297; v8, AL562860; v9, BC05035. For MBNL2: v1, NM_144778; v2, AF061261; v3, NM_207304. For MBNL3: v1, NM_018388; v2, NM_133486; v3, BC042090; v4, CR624180; v5, AY372211; v6, AK002178. Numbers indicate the length of each protein variant (a.a., amino acid). Exons are drawn approximately to scale, but the intervening genomic sequence is not.

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Fig. 4 Muscleblind proteins regulate alternative splicing and subcellular localization of specific transcripts. (A) Biochemical and/or genetic evidence demonstrates that Muscleblind proteins participate in the terminal differentiation of muscles as either promyogenic (MBNL1 and probably MBNL2; represented as MBNL12) or antimyogenic (MBNL3) factors. A triangle represents changes in gene expression. MBNL and CELF proteins act antagonistically in the use of defined alternative exons by binding to intronic splicing enhancers (promoting exon inclusion; bent arrow) and silencers (promoting exon exclusion; inhibitory bent arrow). These proteins regulate a developmental switch between embryonic and adult splice patterns for a number of pre-mRNAs, including cTNT, IR, Tnnt3 and ClC-1, which is critical for their activity. For example, IR isoforms containing exon 11 have a higher signaling capacity than IR isoforms lacking exon 11, whereas exclusion of cryptic exons from pre-mRNA ClC-1 transcripts is necessary for proper chloride conductance regulation in muscles (Savkur et al., 2001; Charlet et al., 2002b). Dmef2 regulates expression of Drosophila muscleblind in the mesoderm. It is likely that their mammalian orthologs, the MEF-2 proteins, similarly regulate MBNL1 and MBNL2 genes. MBNL3 inhibits the terminal muscle marker Myosin heavy chain (MyHC) and the early myogenic marker myogenin in mouse myoblast cell culture experiments (Squillace et al., 2002). (B) MBNL2 proteins mediate localization of integrin a3 proteins to focal adhesions by binding defined sequences within the integrin a3 3 0 untranslated region (zipcode; Adereth et al., 2005). Because integrin adhesion to the extracellular matrix (ECM) is required for muscle differentiation, for example in mature muscle attachments to tendon cells, this function might be relevant during terminal muscle differentiation. mb, plasma membrane.

of splicing on different transcripts, probably through interactions with associated factors in vivo (Fig. 4A). Overexpression of MBNL1, MBNL2 or MBNL3 promoted exon 5 exclusion in cTNT minigenes, but promoted exon 11 inclusion in IR minigenes. Consistently, small interfering RNA-mediated depletion of MBNL1

showed the opposite effect on cTNT and IR splicing. In order to perform this function, MBNL1 binds to a common intronic motif near the alternative cTNT exon 5 characterized by the consensus sequence YGCU(U/ G)Y (where Y is any pyrimidine). CUG-BP (also called CUG-BP1 or BRUNOL2) and embryonic lethal abnormal vision-type RNA-binding protein 3 (ETR-3), belonging to the CELF family of proteins, antagonize MBNL function in exon choice control on human and chicken cTNT and human IR (Philips et al., 1998; Ladd et al., 2001; Savkur et al., 2001; Charlet et al., 2002a; Dansithong et al., 2005). CELF and MBNL proteins bind to distinct cis-elements in cTNT and IR minigenes, thus ruling out a simple competition model for binding as basis for their antagonism. Indeed, minigenes carrying CELF- or MBNL-binding site mutations still responded to the antagonist protein, thus suggesting that both splicing factor families regulate exon choice independently from one another (Ho et al., 2004). Studies with primary cardiomyocyte cultures further substantiate the antagonistic activity of MBNL and CELF proteins in the developmentally regulated alternative splicing of cTNT exon 5 (Ladd et al., 2005). Exon 5 is included in embryonic cTNT transcripts but not in adult heart. In this study, CELF family members CUG-BP and ETR-3 factors promoted exon 5 inclusion, whereas polypyrimidine tract-binding protein (PTB) and MBNL1 proteins repressed inclusion. The developmental switch depended on the regulated expression of the exon 5 inclusion activators and repressors. While CUG-BP and ETR-3 proteins were strongly down-regulated in adult hearts, PTB and MBNL1 expression was maintained throughout heart development. Thus, a balance between positive and negative regulators of exon 5 inclusion determines this developmental change in splicing (Ladd et al., 2005). Consistent with the regulatory role that Muscleblind proteins have on the alternative splicing of muscle transcripts, Drosophila and mouse loss-of-function mutants show a clear musculature phenotype (Artero et al., 1998; Kanadia et al., 2003a). In Drosophila, strong hypomorphic alleles of muscleblind are embryonic lethal. Mutants die as first instar larvae unable to break the chorion and hatch. These larvae are severely paralyzed and their segments are contracted, especially in the abdominal region. Ultrastructural studies revealed that the regular organization of the contractile apparatus (sarcomeres) was compromised. Thick and thin filaments were correctly oriented, but less ordered and densely packed than in wild type. I-bands and mesh-like matrix of Z-bands were absent. Because loss of I-bands occurs naturally when a wild-type muscle is supercontracted, muscleblind mutant embryos might be arrested in a supercontracted state. Terminal muscle differentiation of muscleblind mutant embryos is defective at an additional level. Wild-type muscles in embryos connect to the epidermal cells via either direct or indirect muscle

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attachments (for a review see Brown, 2000). At indirect muscle attachments, many muscle tips converge onto a limited amount of epidermal surface, to which they attach via an accumulation of extracellular matrix known as tendon matrix. In muscleblind mutant embryos, the tendon matrix is strongly reduced and indirect muscle attachments fail to assembly correctly (Artero et al., 1998). In mice, disruption of Mbnl1 leads to a muscle phenotype as well. Mbnl1 mutant animals display overt myotonia, i.e., delayed muscle relaxation because of repetitive action potentials in myofibers, beginning around 6 weeks of age. Immunofluorescence analysis confirmed that a major reduction of chloride channel 1 protein in Mbnl1 mutant animals, because of the splicing abnormalities described above, was the molecular basis for the myotonia. Histological analysis of Mbnl1 mutant mice showed no major degeneration of muscle fibers, but abnormal features included an increase in nuclei with central (abnormal) position and splitting of myofibers (Kanadia et al., 2003a). As expected for a pro-myogenic factor, muscleblind expression becomes activated during Drosophila and mammalian myoblast differentiation. In Drosophila embryos, anti-Muscleblind antibodies detect Muscleblind protein in the nucleus of myoblasts from mid (stage 11) to late embryogenesis, when it is strongly expressed in several mesodermal derivatives, such as the alary (segmentally repeated pairs of ligament cells that anchor the heart to the epidermis), pharyngeal, visceral and somatic (body wall) musculature. Interestingly, Muscleblind is not expressed in the heart or the fat body (homologous to the mammalian liver), two tissues that express MBNL1 and MBNL2 proteins in adult mammals (see below; Artero et al., 1998; Fardaei et al., 2002). Northern blot analysis and cell culture experiments established expression of MBNL genes in several human tissue types (Miller et al., 2000; Fardaei et al., 2002; Squillace et al., 2002). MBNL1 and MBNL2 showed expression in brain, kidney, liver, pancreas and muscle cell types, but while MBNL2 was found in similar levels in all tissues, MBNL1 was more abundant in skeletal muscle and heart. MBNL3 transcripts, however, were expressed at much lower levels, with peak expression in placenta and no expression in skeletal muscle (Fardaei et al., 2002; Squillace et al., 2002). In contrast to human, expression of mouse Mbnl1, Mbnl2 and Mbnl3 genes in adults was more uniform across tissues, although Mbnl1 transcript levels were highest in heart and lowest in skeletal muscles. During embryonic development, expression patterns of Mbnl genes were examined using whole mount in situ hybridization. While all three Mbnl genes were expressed prominently in the developing head region and forelimb bud by 9.5 dpc, later in development the expression patterns of Mbnl1 and Mbnl2 were somehow similar (tongue, mandibular and maxilary regions, lips, thymus, lung and intestines among other tissues). Mbnl3 expression was

stronger in the thymus, lung and intestines (Kanadia et al., 2003b). Given the differences in the expression pattern between MBNL1/MBNL2 and MBNL3, specifically the lack of MBNL3 expression in mature muscle tissues, it is not surprising that MBNL3 inhibited muscle differentiation in cell culture experiments (Squillace et al., 2002). As MBNL3 levels decreased in differentiating MM14 and C2C12 myoblasts, MBNL1 expression activated upon induction of myoblast differentiation of the murine C2C12 cell line (Miller et al., 2000; Squillace et al., 2002). Conversely, MBNL3 overexpression inhibited muscle-specific genes such as myogenin and myosin heavy chain, whereas Mbnl1 knockout mice and muscleblind mutant flies showed terminal muscle differentiation defects (Squillace et al., 2002). Induction of the cell cycle inhibitor p21 marks myoblast withdrawal from the proliferative cell cycle and commitment to become muscle tissue. MBNL3 inhibition of myogenesis, however, is p21-independent, since levels of p21 were unaffected by MBNL3 expression (Squillace et al., 2002). The molecular mechanism by which MBNL3 prevents muscle differentiation is likely to involve regulated splicing of muscle-specific transcripts, since all three MBNL proteins similarly regulate splicing of human cTNT and IR minigenes (Ho et al., 2004, see above). Thus, speculative models would be that MBNL1 and MBNL3 have opposing splice site selection activities on specific transcripts, leading to inclusion or exclusion of exons critically important to the myogenic activity of the protein encoded, and that each MBNL protein binds and regulates different subsets of transcripts. The regulation of these complex tissue-specific patterns of muscleblind expression is largely unknown. Work in Drosophila identified Dmef2, a member of the Myocyte enhancer factor 2 (MEF-2) family of transcription factors implicated in the activation of muscle specific gene expression, as a muscleblind transcriptional regulator (Artero et al., 1998). Such regulation is consistent with the expression of muscleblind during myogenesis and its genetic requirement for terminal muscle differentiation.

An RNA-dependent protein localization mechanism mediated by MBNL2 proteins MBNL proteins are functionally redundant in regulating splicing of transiently expressed cTNT and IR minigenes in skeletal muscle cultures (Ho et al., 2004). A recent report, however, identified MBNL2 as an RNAbinding protein that co-localizes with integrin a3 mRNA in the cytoplasm, which leads to its cognate protein to concentrate on focal adhesion complexes (Adereth et al., 2005). MBNL2-specific small interfering RNA-treated carcinoma cells localized integrin a3

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protein mainly in the cell body, instead of in adhesion plaques where it normally co-localizes with paxilin in the migration fronts. These results implicate MBNL2 as an essential function for integrin a3 protein localization (Fig. 4B). MBNL2 proteins are mainly expressed in or near large cytoplasmic plaques that also contain phospho focal adhesion kinase (pFAK). This localization is microtubule dependent. To control the subcellular localization of integrin a3 proteins, MBNL2 physically associates with a small sequence motif termed zipcode (ACACCC) within the 3 0 untranslated region of the integrin a3 mRNA. The binding is not only necessary, but sufficient to target the GFP protein encoded by a chimeric GFP-integrin a3 zipcode mRNA to adhesion plaques in an MBNL2-dependent fashion. Overall, it has been suggested that MBNL2 is an integrin a3 RNA transporter from the nucleus to defined cytoplasmic sites, where integrin a3 proteins are ultimately made and incorporated into adhesion complexes (Adereth et al., 2005). In addition, integrin a3, non-muscle b-actin, and macrophage colony stimulating factor 1 receptor (cfms) transcripts were found up-regulated by MBNL2 in a limited cDNA array analysis for structure- and motility-related genes. MBNL2 positive regulation of integrin a3 expression was post-transcriptional, although the authors did not address whether MBNL2 controlled integrin a3 pre-mRNA splicing. As shown for integrin a3, MBNL2 might similarly regulate b-actin and c-fms expression post-transcriptionally, as well as the subcellular localization of the proteins encoded. In light of these results, muscleblind loss-of-function mutations in both mouse and Drosophila deserve further exploration. Some aspects of the phenotype of Drosophila muscleblind mutants are not satisfactorily explained just invoking missplicing events. Tendon matrix deposition at muscle attachment sites requires muscleblind function at growing muscle tips, thus suggesting control over the subcellular location of a Muscleblindfunction target protein. Integrins are known to mediate cell–cell and cell–substrate recognition, and hence are prime candidates. Furthermore, muscleblind and integrin loss-of-function phenotypes are similar in some aspects (Artero et al., 1998). Although it is possible that a missplicing event could interfere with a localized protein activity, evidence suggesting that MBNL2 localizes integrin a3 protein to focal adhesion complexes provides an alternative explanation. These results identify a new RNA-dependent protein localization mechanism mediated by MBNL2. Lack of a homologous function in Drosophila may help explain cell structure and adhesion changes, such as lack of rhabdomeres, the light harvesting structures of photoreceptors, in muscleblind mutant retina clones (see the next section), and absence of tendon matrix at muscle attachment sites. Interestingly, some combinations of hypomorphic muscleblind loss-of-function mutations allow adult escapers to hatch, thus revealing later requirements of muscleblind

function during development. These escapers have wing blisters, a phenotype characteristic of integrin mutants in Drosophila, as well as wing venation defects or unexpanded wings (Prokopenko et al., 2000).

Muscleblind is required in the differentiation of several cell types A genetic screen published 10 years ago identified muscleblind as a gene required for the morphogenesis of chordotonal organs during the development of the Drosophila embryonic peripheral nervous system (Kania et al., 1995). The chordotonal organs, located in the body wall, function as propioreceptors and are composed of a neuron and three support cells. muscleblind mutant embryos display lateral chordotonal neurons that are associated more closely than in wild type, exhibit a mild loss of neurons, and show abnormal neuronal morphology and fasciculation defects. This phenotype suggests that muscleblind is essential during the terminal steps of neuron differentiation and morphogenesis (Kania et al., 1995; Prokopenko et al., 2000). A role for muscleblind during the development of central nervous system neurons is also likely, since the Muscleblind protein is detected in a segmentally repeated pattern of cells in the central nervous system of the Drosophila embryo (Artero et al., 1998). Muscleblind proteins are also pivotal to the differentiation of Drosophila eye photoreceptors. In muscleblind mutant mitotic clones, the initial steps of photoreceptor induction and determination are normal as judged from the expression of neural (Elav protein) and photoreceptor-specific markers in eye imaginal discs. Proper morphogenesis of photoreceptor neurons, however, fails. Tangential sections through adult eye muscleblind clones reveal that the rhabdomeres are smaller in diameter, are malformed, and frequently do not extend into basal retinal regions in hypomorphic alleles. In muscleblind null alleles, rhabdomeres are lacking altogether (Begemann et al., 1997). Overexpression of Drosophila muscleblind protein isoforms A and C in the developing retina, however, does not interfere with rhabdomere morphogenesis but leads to typical planar cell polarity defects, which are stronger and combine with photoreceptors losses when MblC is overexpressed (Garcia-Casado et al., 2002). Interestingly, Mbnl1 knockout mice develop distinctive ocular cataracts (Kanadia et al., 2003a) even though, for an accurate comparison, the retinas of these mutant mice would have to be analyzed. Worth noting is the resemblance of the phenotype of muscleblind mutant eye clones and the loss-of-function phenotype of mutations in crumbs, a central regulator of the epithelial apical-basal polarity in Drosophila (Izaddoost et al., 2002), which might underlie a functional relationship between both processes.

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Two studies have found MBNL proteins to be differentially expressed when comparing differentiated versus undifferentiated cell types. The first study reports the results of a suppression subtractive hybridization technique that compared transcripts expressed by murine 3T3-F442A preadipocytes before and after growth hormone-dependent adipocyte differentiation (Shang et al., 2002). This study identified MBNL2 as a transcript that was rapidly up-regulated by growth hormones in 3T3-F442A pre-adipocytes as these cells become primed for terminal differentiation. Just as muscleblind is an important RNA-binding protein involved in Drosophila muscle and neural development, it may play a similar role in vertebrate adipogenesis. In the second study, a subtracted complementary DNA library from highly purified murine fetal liver stem cells identified mouse MBNL3 transcripts in hematopoietic stem cells (Phillips et al., 2000). Because the hallmark property of stem cells is their ability to balance selfrenewal and commitment to differentiation, MBNL3 may act by inhibiting terminal blood cell differentiation. This is remarkably similar to the roles played by MBNL1 and MBNL3 proteins during muscle differentiation, where the balance between their pro (MBNL1) and anti-myogenic (MBNL3) activities was proposed to determine the proper execution of the myogenic program (Miller et al., 2000; Squillace et al., 2002).

Muscleblind proteins are key targets in RNA-mediated pathogenesis Because loss of muscleblind function in both Drosophila and mouse results in strong phenotypes, including lethality, it is not surprising that human MBNL proteins are linked with hereditary disorders, most importantly myotonic dystrophies (DM). These are autosomal dominant neuromuscular diseases associated with CTG repeat expansions in the 3 0 -untranslated region of the DM protein kinase (DMPK) gene (DM1), and intronic CCTG repeat expansions in the ZNF9 gene (DM2). Both DMPK and ZNF9 mutant transcripts accumulate in distinctive foci within muscle nuclei. DMs are multisystemic diseases, although characteristic features are primarily muscular, such as myotonia (muscle hyperexcitability) and muscle wasting. Other abnormalities include distinctive cataracts, insulin resistance and cognitive impairment. Because of shared clinical symptoms between DM1 and DM2, a common pathophysiologic mechanism has been proposed (for recent reviews see Nykamp and Swanson, 2004; Ranum and Day, 2004; Day and Ranum, 2005). Work by the group of M. Swanson first shed light as to how non-coding CTG trinucleotide expansions were pathogenic to DM1 patients (Miller et al., 2000). In this study, MBNL proteins were isolated from HeLa cell

nuclear extracts by selectively associating with large CUG expansions (420 repeat units, forming RNA hairpins; Fig. 5B). Importantly, the interaction was direct, since purified His-tagged MBNL1 protein alone bound and crosslinked to dsCUG RNAs in vitro. MBNL1 proteins also accumulated in multiple nuclear foci (Miller et al., 2000). The group of Ishiura further extended these observations using various synthetic RNAs in a yeast three-hybrid assay (Kino et al., 2004). MBNL1 was shown to specifically interact with CHHG and CHG repeats (where H is A, U or C), which include those found in DMPK (CUG; DM1) and ZNF9 (CCTG; DM2) mutant transcripts. Interestingly, MBNL1 did not interact with a genuine double-stranded RNA comprising CAG/CUG repeats, suggesting that MBNL1 prefers bulge-containing double-stranded RNAs. Deletion analysis of MBNL1 showed that deletions of the C-terminus and flanking alanine-rich region cause no reduction in the RNA-binding activity of the protein, whereas deletions including each zinc finger domain suggest that all four zinc fingers are necessary for full RNA-binding activity. Overall, these results support a toxic RNA model in which the known nuclear accumulation of mutant DMPK transcripts in DM1 myoblasts would lead to aberrant recruitment of MBNL proteins and subsequent deleterious effects on cellular differentiation. Additional support to the toxic RNA model comes from co-localization studies, both in cell culture and tissue samples, demonstrating that MBNL1, MBNL2 and MBNL3 proteins localize to nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells (Fardaei et al., 2001; Mankodi et al., 2001; Fardaei et al., 2002). Thus, in vivo, mutant DMPK and ZNF9 transcripts aberrantly sequester MBNL proteins in the nucleus. Such sequestration might induce MBNL loss-of-function phenotypes by diverting available MBNL proteins from their normal molecular function (Fig. 5A,5B,5D). Genetic studies support that specific muscle, eye and RNA splicing abnormalities that are characteristic of DM pathology stem from MBNL lossof-function. Mice carrying a targeted deletion of the exon containing the major initiation codon in Mbnl1 (Mbnl1 knockouts) display overt myotonia, distinctive ocular cataracts and histologic abnormalities in muscles that are similar to those typical of DM1 patients. Moreover, Mbnl1 mutant mice showed abnormal regulation of alternative splicing of cTNT, Clcn-1 and Tnnt3 that mimicked splicing alterations found in DM patients (Kanadia et al., 2003a). In all cases, the developmental switch between embryonic/fetal to adult pattern of alternative splicing failed to take place (Philips et al., 1998; Charlet et al., 2002b; Kanadia et al., 2003a). Thus, loss of specific Mbnl1 protein isoforms, those containing exon 3 (Fig. 3), is sufficient to cause DM1-like phenotypes. Although evolutionarily distant, the study of muscleblind loss-of-function

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Fig. 5 Implication of MBNL proteins in DM1 foci formation and RNA-mediated pathogenesis. (A) In normal cells, MBNL proteins accumulate in the nucleus where they regulate alternative splicing of defined transcripts by binding to specific RNA-binding sites. CELF proteins antagonize the activity of MBNL proteins. Choice of exons labeled with the words MES, SEN and GER represents normal splicing. In some cell types (normal human myoblasts and murine myotubes are examples) MBNL1 proteins are detected in both the cytoplasm and the nucleus, thus suggesting a role for MBNL proteins outside the nucleus (Miller et al., 2000). Indeed, MBNL2 proteins localized specific mRNAs in the cytoplasm (Adereth et al., 2005). (B) Large CUG-repeat expansions are toxic to the cell and lead to characteristic pre-mRNA splicing alterations and ribonuclear foci formation. According to a model (Houseley et al., 2005), MBNL and nuclear matrix-binding proteins PSF/p54nrb

interact. This interaction might provide a physical attachment for ribonuclear foci as explained in the text. (C) In addition to MBNL, repeat expansion-containing transcripts recruit RNA-binding proteins (Staufen, hnRNPF, hnRNPH and Spen) and transcription factors (Sp1, RARg, STAT3 and STAT1; see text for details). Redistribution of transcription factors to DM1 foci reduces expression of defined genes, as for example the Sp1 transcriptional target gene ClC-1 (Ebralidze et al., 2004). (D) Defined splicing alterations in DM patients lead to abnormal protein isoforms including those for ClC-1, IR and cTNT, whose aberrant molecular function explains DM symptoms such as myotonia (ClC-1), insulin resistance (IR) and, potentially, cardiac abnormalities (cTNT). MBNL depletion alone, however, does not trigger such defined missplicing events (Ho et al., 2005), thus suggesting additional steps in the pathogenetic pathway.

mutations in Drosophila provides striking examples of DM-like phenotypes. muscleblind mutant embryos show the absence of sarcomeric Z-bands, as similarly described in DM1 patients (Ludatscher et al., 1978; Artero et al., 1998), and hypercontracted abdomen that might be equivalent to human myotonia. Genetic elimination of muscleblind function in developing Drosophila eyes interferes with photoreceptor differentiation (Begemann et al., 1997), which might induce photoreceptor degeneration as described for some DM1 patients (Harper, 2001). Since its initial description (Taneja et al., 1995) the molecular composition of DM1 foci has been under intense investigation. Biochemical and genetic studies have demonstrated that besides MBNL proteins other nuclear factors also colocalize to ribonuclear foci (Fig. 5C; Miller et al., 2000; Fardaei et al., 2001; Mankodi et al., 2001; Fardaei et al., 2002; Mankodi et al., 2003;

Jiang et al., 2004). Additional components of DM1 foci are the RNA-binding proteins NonA (the Drosophila PSF/p54nrb ortholog; Houseley et al., 2005), hnRNP H and hnRNP F (Jiang et al., 2004; Kim et al., 2005). Furthermore, genetic studies in Drosophila suggest that the RNA-binding proteins Staufen, Split ends and CG3249 may interact with expanded CUG repeat sequences in vivo (Mutsuddi et al., 2004). Specific transcription factors also redistribute to ribonuclear foci (Ebralidze et al., 2004). Mutant RNA binds and sequesters transcription factors such as Specificity protein 1 (Sp1), signal transducer and activator of transcription family members STAT1 and STAT3, and retinoic acid receptor gamma (RARg). Diverse genes are consequently reduced in expression, including the human chloride channel 1 (ClC-1), the transcriptional target of Sp1. Thus, DM1 foci are complex structures made up of mutant DMPK RNA, RNA-binding proteins (including

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Muscleblind proteins), transcription factors and possibly other protein partners. Considerable effort has been devoted to understanding whether foci formation in DM1 cells is pathogenic, protective, or perhaps irrelevant. Recent cell culture experiments indicate that both CUG and CAG (as control) repeats form RNA foci that colocalize with cotransfected MBNL1_v1 and endogenous MBNL1_v1,3 proteins. However, only CUG repeats altered splicing of cTNT and IR, the two pre-mRNAs known to be altered in DM1 patients that were tested (Philips et al., 1998; Savkur et al., 2001). Therefore, disruption of splicing of MBNL1 pre-mRNA targets can be uncoupled from the accumulation of MBNL1 into nuclear foci, and ribonuclear foci formation was concluded not to be pathogenic per se (Ho et al., 2005). These results, though, are limited by the technical impossibility of detecting all MBNL1 protein isoforms in the RNA foci formed. It is formally possible that CUG and CAG repeat expansions sequester some MBNL1 protein isoforms (such as MBNL1_v1,3), but additional protein isoforms bind differentially to CAG and CUG repeat expansions. Likewise, a Drosophila model of DM1 expressing 162 CUG repeats in muscle cells (using the Gal4/UAS system for targeted tissue expression) readily sequestered Muscleblind proteins into ribonuclear foci but showed no obvious phenotype (Houseley et al., 2005). Taken together, these results suggest that the toxic effect brought about by CUG-repeat expansion RNAs involves more than Muscleblind sequestration alone. A first hint into the mechanism by which expression of expanded CUG repeats is toxic to cells comes from co-transfection experiments using chicken skeletal muscle cultures and cTNT and IR minigenes as the functional assay of MBNL activity. In these experiments, wild-type human cTNT minigenes containing MBNL1and CUG-BP binding sites flanking alternative exon 5 responded to coexpressed CUG repeat RNA by favouring exon 5 inclusion, as similarly found by small interfering RNA MBNL1 depletion. CUG-BP mutant human cTNT minigenes, however, do not respond to coexpressed CUG repeat RNA and include exon 5-like controls, suggesting that an intact CUG-BP-binding site mediates the trans-dominant effect exerted by expanded CUG repeats over the alternative splicing of specific transcripts (Ho et al., 2004). One working model proposes that MBNL and CELF protein families mediate developmentally regulated splicing programs, and expression of CUG or CCUG repeats either initiates an aberrant signaling event or interferes with a signaling cascade for which MBNL and CELF proteins are downstream mediators (Ho et al., 2005). As mentioned above, Muscleblind family members contain highly conserved putative phosphorylation sites, thus making them likely to be functionally relevant (Fig. 3). In an attempt to integrate these results with previous observations, we propose that aberrant recruitment of

MBNL proteins to large repeat expansion RNAs reduces available MBNL proteins and sensitizes the cell to additional molecular alterations that impinge on MBNL function. Sources of supplementary molecular changes might be downregulation of MBNL target RNA transcripts (such as ClC-1 pre-mRNAs), because of transcription factor leaching, and interference with intracellular signalling pathways that regulate MBNL function, as might be recruitment of signal transductors to DM1 foci (Fig. 5). Furthermore, it was recently hypothesized that CUG hairpins might become a source of microRNAs and/or small interfering RNAs that would silence CTG repeat expansion-containing genes. Because MBNL1 involves a polyalanine sequence (Fig. 2), which is encoded in the mRNA by a nearcomplementary sequence to the repeated CUG triplet sequence of the suggested small interfering RNA, it might be a molecular target for a lower, but significant, RNA silencing (Malinina, 2005). Irrespective of its detailed mechanism, MBNL loss-of-function and CUGBP gain-of-function may alter developmentally programmed alternative splicing of pre-mRNAs in DM patients. It is currently unclear whether a single protein plays a predominant role in the maintenance of foci integrity or whether a complex interaction of RNAs and proteins that bind to CUG repeat expansions is necessary to trap the mutant DMPK RNA into the aggregates observed in DM1 cells. Obvious candidates are Muscleblind proteins themselves. Indeed, sequestration of MBNL1 by the expanded CUG repeats is the primary determinant of DM1 focus formation in cell culture experiments. Small interfering RNA-mediated down-regulation of MBNL1 in DM1 myoblast results in an approximately 70% reduction of DM1 foci, whereas down-regulation of MBNL2 alone or CUG-BP resulted in a small reduction in the number of foci (approximately 25% and 20% respectively). Thus, these experiments show that depletion of MBNL1 alone is sufficient to abolish the great majority of DM1 foci in vivo (Dansithong et al., 2005). A Drosophila model for CUG-mediated cell toxicity, however, showed that DM1 foci formed in most cell types in the absence of detectable Muscleblind. Furthermore, expression of Muscleblind protein isoforms in tissues in which it was normally absent was unable to mediate ribonuclear foci formation. Thus, Muscleblind is neither necessary nor sufficient to mediate ribonuclear foci formation of expanded CUG repeat RNAs in Drosophila (Houseley et al., 2005). Ribonuclear foci in these model flies co-localized with NonA, a protein whose mammalian orthologs PSF and p54nrb were found to associate with matrin 3 and to mediate attachment of dsRNA to the nuclear matrix (Zhang and Carmichael, 2001). This fact provides a potential physical link between expanded CUG repeat RNA and an immobile nuclear protein network, the nuclear matrix. According to a model put forward by

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Houseley et al. (2005), transcripts containing short (CUG)n tracts in normal cells would bind to a single MBNL protein and weakly associate with the nuclear matrix via PSF/p54nrb (Fig. 5B). Because only a few interactions are established, matrix-bound and freely diffusible MBNL proteins would co-exist. Large expanded (CUG)n repeat tracts, on the other hand, are expected to bind several molecules of MBNL. Multiple MBNL proteins would need to dissociate simultaneously in order to release matrix-bound CUG repeat expansions. Delayed dissociation and additional binding of MBNL proteins to vacant binding sites in the expanded (CUG)n tract would ultimately develop into the large RNA-protein aggregates observed in DM1 cells (Houseley et al., 2005). Genetic evidence in Drosophila implicates Muscleblind proteins in Spinocerebellar Ataxia 8 (SCA8), a dominant disorder caused by CTG repeat expansion in the non-coding SCA8 transcripts. Despite the molecular similarities between the DM and SCA8 mutations, SCA8 causes a central nervous system disease without the multisystemic features of DM. A Drosophila model, in which normal and mutant human SCA8 transcripts were expressed in the retina, exhibited late-onset and progressive neurodegeneration, as similarly found in SCA8 patients. muscleblind loss-of-function mutations dominantly enhanced the neurodegenerative phenotype in these model flies, thus also implicating Muscleblind proteins in the SCA8 pathology (Mutsuddi et al., 2004). In summary, Muscleblind proteins may be relevant to human pathologies involving toxic non-coding repeat expansions of the type CHHG or CHG (H 5 A,U,C), to which they aberrantly bind (Kino et al., 2004), thus offering a common component in their pathogenic mechanism.

Perspectives and future directions Despite great advances in understanding the molecular functions of Muscleblind proteins, several questions remain unanswered. We know very little about the mechanism by which Muscleblind proteins regulate alternative splicing. Muscleblind and CELF proteins are antagonistic in splice site selection during cTNT and IR pre-mRNA splicing, two direct targets, but their activities are independent (Ho et al., 2004; Ladd et al., 2005). PTB and MBNL proteins are repressors of exon 5 inclusion in cTNT pre-mRNA splicing in cardiomyocytes, but it is unknown whether they physically interact (Ladd et al., 2005). In order to understand this regulatory mechanism controlling splice site selection, one aspect that needs clarification is how can Muscleblind and CELF proteins activate splicing of one substrate and repress splicing of another. It remains to be seen whether antagonistic regulation by MBNL and CELF

family members is mechanistically linked, or some premRNAs are targets of just one family of splice regulators (Ho et al., 2004). Furthermore, whereas MBNL1 is expressed during mammalian myogenesis (Miller et al., 2000), MBNL3 overexpression in myoblasts inhibits muscle differentiation (Squillace et al., 2002). Although all three MBNL proteins behave similarly in cTNT and IR splice assays (Ho et al., 2004), MBNL3 must control alternative splicing of specific premRNAs, encoding for antimyogenic factors that mediate its inhibitory effect. Alternatively, MBNL3 might have molecular functions other than splice regulation. Defining Muscleblind protein partners, functional domains involved in splice site regulation and additional pre-mRNA targets would provide a way to answer these important questions. It is an established fact that mRNA export and premRNA splicing are functionally coupled (reviewed in Reed and Hurt, 2002; Reed, 2003). oskar messenger RNA localization at the posterior pole of the Drosophila oocyte provides a remarkable example. Recent work showed that splicing at the first exon–exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. oskar 3 0 UTR, considered sufficient for posterior localization of unrelated transcripts in oocytes, actually required endogenous oskar mRNA for subcellular targeting (Hachet and Ephrussi, 2004). MBNL2 regulation of integrin a3 messenger ribonucleoprotein complex assembly in the cytoplasm, and protein localization in adhesion plaques, may be similarly coupled to integrin a3 pre-mRNA splicing. Indeed, MBNL2 proteins can regulate splicing of transiently expressed minigenes in cell culture (Ho et al., 2004). Conversely, it remains to be seen whether MBNL1 and MBNL3 proteins similarly participate in the export of specific mRNAs from the nucleus to the cytoplasm. The study of the molecular pathogenesis of DM (and SCA8 more recently) has provided several key observations to elucidate the molecular function of Muscleblind proteins. There is no doubt that these studies will contribute new target pre-mRNAs for MBNL proteins as well as additional details of their molecular roles in normal and diseased states. An outstanding question is whether MBNL proteins are involved in the elaboration of other human disorders. Some reports have associated MBNL proteins with the cancerous state, even though the evidence is fragmentary and correlative at best. Many tumors, including lung and colorectal carcinomas, expressed MBNL2 differentially. Moreover, MBNL2 proteins regulated integrin a3 protein localization in adhesion plaques. Integrin a3b1 dimer is a major pro-migratory adhesion molecule in metastatic cells, thus suggesting a potential role for MBNL2 in the metastatic transformation (Adereth et al., 2005). MBNL1 was a partner of the BCL6 translocation observed in follicular lymphomas (Akasaka et al., 2003).

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Clearly, additional work is necessary to elucidate the potential anti-tumorigenic function of MBNL proteins in normal cells, but this involvement is likely given the important developmental roles played by MBNL proteins in controlling terminal differentiation of a number of cell types. Over the last few years, studies aimed to understand DM pathogenesis and the molecular function of Muscleblind proteins converged into a common theme. Modulation of Muscleblind protein levels, and their antagonistic CELF proteins, acts as a developmental ‘‘switch’’ to coordinate the alternative splicing of different pre-mRNAs (Ladd et al., 2005). Toxic CUG repeat expansion RNAs interfere with such developmental switches, thus leading to defined missplicing events ultimately responsible for specific DM symptoms. Although depletion of Muscleblind proteins by aberrant binding to CUG repeat expansion RNAs provided a plausible hypothesis for toxicity, recent results suggest a more complex mechanism. We hope future work will help untangle the molecular roles that Muscleblind proteins play in the normal and the pathogenic states and pave the way to palliative treatments. Acknowledgments We thank I. Marin, K. Gonzalez, P. Morcillo and N. Paricio for helpful comments on the manuscript. We also thank Dr. Y. Kino for kindly providing the graphics file that served as basis for Fig. 3. Work from the authors 0 laboratory is supported by grants from the Spanish Ministry of Science and Education (MEC; SAF2003-03536) and Generalitat Valenciana (GV2004-B164). M.V. and L.M are supported by fellowships from the MEC and the Muscular Dystrophy Association, respectively. R.A. is an investigator from the Ramon y Cajal program of the MEC.

References Adereth, Y., Dammai, V., Kose, N., Li, R. and Hsu, T. (2005) RNA-dependent integrin a3 protein localisation regulated by the Muscleblind-like protein MLP. Nat Cell Biol 7:1140–1147. Akasaka, T., Lossos, I.S. and Levy, R. (2003) BCL6 gene translocation in follicular lymphoma: a harbinger of eventual transformation to diffuse aggressive lymphoma. Blood 102: 1443–1448. Artero, R., Prokop, A., Paricio, N., Begemann, G., Pueyo, I., Mlodzik, M., Perez-Alonso, M. and Baylies, M.K. (1998) The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. Dev Biol 195:131–143. Begemann, G., Paricio, N., Artero, R., Kiss, I., Perez-Alonso, M. and Mlodzik, M. (1997) muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins. Development 124:4321–4331. Black, D.L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336. Blackshear, P.J. (2002) Tristetraprolin and other CCCH tandem zinc-finger proteins in the regulation of mRNA turnover. Biochem Soc Trans 30:945–952. Brown, N.H. (2000) Cell–cell adhesion via the ECM: integrin genetics in fly and worm. Matrix Biol 19:191–201. Brown, R.S. (2005) Zinc finger proteins: getting a grip on RNA. Curr Opin Struct Biol 15:94–98.

Cartegni, L., Chew, S.L. and Krainer, A.R. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3:285–298. Cooper, T.A. and Ordahl, C.P. (1985) A single cardiac troponin T gene generates embryonic and adult isoforms via developmentally regulated alternate splicing. J Biol Chem 260:11140–11148. Charlet, B.N., Logan, P., Singh, G. and Cooper, T.A. (2002a) Dynamic antagonism between ETR-3 and PTB regulates cell type-specific alternative splicing. Mol Cell 9:649–658. Charlet, B.N., Savkur, R.S., Singh, G., Philips, A.V., Grice, E.A. and Cooper, T.A. (2002b) Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 10:45–53. Dansithong, W., Paul, S., Comai, L. and Reddy, S. (2005) MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1. J Biol Chem 280:5773–5780. Day, J.W. and Ranum, L.P. (2005) RNA pathogenesis of the myotonic dystrophies. Neuromuscul Disord 15:5–16. Ebralidze, A., Wang, Y., Petkova, V., Ebralidse, K. and Junghans, R.P. (2004) RNA leaching of transcription factors disrupts transcription in myotonic dystrophy. Science 303:383–387. Fardaei, M., Larkin, K., Brook, J.D. and Hamshere, M.G. (2001) In vivo co-localisation of MBNL protein with DMPK expandedrepeat transcripts. Nucleic Acids Res 29:2766–2771. Fardaei, M., Rogers, M.T., Thorpe, H.M., Larkin, K., Hamshere, M.G., Harper, P.S. and Brook, J.D. (2002) Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 11:805–814. Faustino, N.A. and Cooper, T.A. (2003) Pre-mRNA splicing and human disease. Genes Dev 17:419–437. Garcia-Casado, M.Z., Artero, R.D., Paricio, N., Terol, J. and Perez-Alonso, M. (2002) Generation of GAL4-responsive muscleblind constructs. Genesis 34:111–114. Gomperts, M., Pascall, J.C. and Brown, K.D. (1990) The nucleotide sequence of a cDNA encoding an EGF-inducible gene indicates the existence of a new family of mitogen-induced genes. Oncogene 5:1081–1083. Grabowski, P.J. and Black, D.L. (2001) Alternative RNA splicing in the nervous system. Prog Neurobiol 65:289–308. Hachet, O. and Ephrussi, A. (2004) Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization. Nature 428:959–963. Harper, P. (2001) Myotonic dystrophy. London: Saunders. Ho, T.H., Charlet, B.N., Poulos, M.G., Singh, G., Swanson, M.S. and Cooper, T.A. (2004) Muscleblind proteins regulate alternative splicing. Embo J 23:3103–3112. Ho, T.H., Savkur, R.S., Poulos, M.G., Mancini, M.A., Swanson, M.S. and Cooper, T.A. (2005) Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy. J Cell Sci 118:2923–2933. Houseley, J.M., Wang, Z., Brock, G.J., Soloway, J., Artero, R., Perez-Alonso, M., O’Dell, K.M. and Monckton, D.G. (2005) Myotonic dystrophy associated expanded CUG repeat muscleblind positive ribonuclear foci are not toxic to Drosophila. Hum Mol Genet 14:873–883. Hudson, B.P., Martinez-Yamout, M.A., Dyson, H.J. and Wright, P.E. (2004) Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d. Nat Struct Mol Biol 11: 257–264. Izaddoost, S., Nam, S.C., Bhat, M.A., Bellen, H.J. and Choi, K.W. (2002) Drosophila crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416:178–183. Jiang, H., Mankodi, A., Swanson, M.S., Moxley, R.T. and Thornton, C.A. (2004) Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet 13:3079–3088. Kanadia, R.N., Johnstone, K.A., Mankodi, A., Lungu, C., Thornton, C.A., Esson, D., Timmers, A.M., Hauswirth, W.W. and

80 Swanson, M.S. (2003a) A muscleblind knockout model for myotonic dystrophy. Science 302:1978–1980. Kanadia, R.N., Urbinati, C.R., Crusselle, V.J., Luo, D., Lee, Y.J., Harrison, J.K., Oh, S.P. and Swanson, M.S. (2003b) Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3. Gene Expr Patterns 3:459–462. Kania, A., Salzberg, A., Bhat, M., D’Evelyn, D., He, Y., Kiss, I. and Bellen, H.J. (1995) P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster. Genetics 139:1663–1678. Kay, B.K., Williamson, M.P. and Sudol, M. (2000) The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. Faseb J 14:231–241. Kim, D.H., Langlois, M.A., Lee, K.B., Riggs, A.D., Puymirat, J. and Rossi, J.J. (2005) HnRNP H inhibits nuclear export of mRNA containing expanded CUG repeats and a distal branch point sequence. Nucleic Acids Res 33:3866–3874. Kino, Y., Mori, D., Oma, Y., Takeshita, Y., Sasagawa, N. and Ishiura, S. (2004) Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats. Hum Mol Genet 13:495–507. Ladd, A.N., Charlet, N. and Cooper, T.A. (2001) The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol 21: 1285–1296. Ladd, A.N., Stenberg, M.G., Swanson, M.S. and Cooper, T.A. (2005) Dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development. Dev Dyn 233:783–793. Lai, W.S., Carballo, E., Strum, J.R., Kennington, E.A., Phillips, R.S. and Blackshear, P.J. (1999) Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol 19:4311–4323. Lai, W.S., Kennington, E.A. and Blackshear, P.J. (2002) Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs. J Biol Chem 277:9606– 9613. Ludatscher, R.M., Kerner, H., Amikam, S. and Gellei, B. (1978) Myotonia dystrophica with heart involvement: an electron microscopic study of skeletal, cardiac, and smooth muscle. J Clin Pathol 31:1057–1064. Malinina, L. (2005) Possible involvement of the RNAi pathway in trinucleotide repeat expansion diseases. J Biomol Struct Dyn 23:233–235. Maniatis, T. and Tasic, B. (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418:236–243. Mankodi, A., Teng-Umnuay, P., Krym, M., Henderson, D., Swanson, M. and Thornton, C.A. (2003) Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 54:760–768. Mankodi, A., Urbinati, C.R., Yuan, Q.P., Moxley, R.T., Sansone, V., Krym, M., Henderson, D., Schalling, M., Swanson, M.S. and Thornton, C.A. (2001) Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Mol Genet 10:2165–2170. Miller, J.W., Urbinati, C.R., Teng-Umnuay, P., Stenberg, M.G., Byrne, B.J., Thornton, C.A. and Swanson, M.S. (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. Embo J 19:4439–4448. Monferrer, L. and Artero, R. (2006) An interspecific functional complementation test in Drosophila for introductory genetics laboratory courses. J Hered 97(1). Musunuru, K. (2003) Cell-specific RNA-binding proteins in human disease. Trends Cardiovasc Med 13:188–195. Mutsuddi, M., Marshall, C.M., Benzow, K.A., Koob, M.D. and Rebay, I. (2004) The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila. Curr Biol 14:302–308.

Nissim-Rafinia, M. and Kerem, B. (2005) The splicing machinery is a genetic modifier of disease severity. Trends Genet 21:480–483. Nykamp, K.R. and Swanson, M.S. (2004) Toxic RNA in the nucleus: unstable microsatellite expression in neuromuscular disease. Prog Mol Subcell Biol 35:57–77. Pelisch, F., Blaustein, M., Kornblihtt, A.R. and Srebrow, A. (2005) Cross-talk between signaling pathways regulates alternative splicing: a novel role for JNK. J Biol Chem 280:25461–25469. Philips, A.V., Timchenko, L.T. and Cooper, T.A. (1998) Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science 280:737–741. Phillips, R.L., Ernst, R.E., Brunk, B., Ivanova, N., Mahan, M.A., Deanehan, J.K., Moore, K.A., Overton, G.C. and Lemischka, I.R. (2000) The genetic program of hematopoietic stem cells. Science 288:1635–1640. Prokopenko, S.N., He, Y., Lu, Y. and Bellen, H.J. (2000) Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins. Genetics 156:1691–1715. Ranum, L.P. and Day, J.W. (2004) Pathogenic RNA repeats: an expanding role in genetic disease. Trends Genet 20:506–512. Reed, R. (2003) Coupling transcription, splicing and mRNA export. Curr Opin Cell Biol 15:326–331. Reed, R. and Hurt, E. (2002) A conserved mRNA export machinery coupled to pre-mRNA splicing. Cell 108:523–531. Savkur, R.S., Philips, A.V. and Cooper, T.A. (2001) Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 29:40–47. Schwerk, C. and Schulze-Osthoff, K. (2005) Regulation of apoptosis by alternative pre-mRNA splicing. Mol Cell 19:1–13. Shang, C.A., Thompson, B.J., Teasdale, R., Brown, R.J. and Waters, M.J. (2002) Genes induced by growth hormone in a model of adipogenic differentiation. Mol Cell Endocrinol 189:213–219. Shin, C. and Manley, J.L. (2004) Cell signalling and the control of pre-mRNA splicing. Nat Rev Mol Cell Biol 5:727–738. Singh, R. (2002) RNA-protein interactions that regulate premRNA splicing. Gene Expr 10:79–92. Squillace, R.M., Chenault, D.M. and Wang, E.H. (2002) Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR. Dev Biol 250:218–230. Stamm, S. (2002) Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome. Hum Mol Genet 11:2409–2416. Taneja, K.L., McCurrach, M., Schalling, M., Housman, D. and Singer, R.H. (1995) Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128:995–1002. Trembley, J.H., Tatsumi, S., Sakashita, E., Loyer, P., Slaughter, C.A., Suzuki, H., Endo, H., Kidd, V.J. and Mayeda, A. (2005) Activation of pre-mRNA splicing by human RNPS1 is regulated by CK2 phosphorylation. Mol Cell Biol 25:1446–1457. Wain, H.M., Bruford, E.A., Lovering, R.C., Lush, M.J., Wright, M.W. and Povey, S. (2002) Guidelines for human gene nomenclature. Genomics 79:464–470. Worthington, M.T., Amann, B.T., Nathans, D. and Berg, J.M. (1996) Metal binding properties and secondary structure of the zinc-binding domain of Nup475. Proc Natl Acad Sci USA 93:13754–13759. Xu, X., Yang, D., Ding, J.H., Wang, W., Chu, P.H., Dalton, N.D., Wang, H.Y., Bermingham, J.R. Jr, Ye, Z., Liu, F., Rosenfeld, M.G., Manley, J.L., Ross, J. Jr., Chen, J., Xiao, R.P., Cheng, H. and Fu, X.D. (2005) ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell 120:59–72. Zhang, F., Barboric, M., Blackwell, T.K. and Peterlin, B.M. (2003) A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb. Genes Dev 17:748–758. Zhang, Z. and Carmichael, G.G. (2001) The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs. Cell 106:465–475.