Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF-and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.

Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSEdependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP-and ETR-3-like factors, specifically bound MSEcontaining RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.The generation of multiple, functionally distinct protein isoforms from a single gene via alternative splicing is a common means of regulating gene expression. It has been estimated that more than one-third of human genes are alternatively spliced (21). Despite the prevalence of alternative splicing, the mechanisms by which it is regulated are not well understood. Cis-acting elements that mediate alternative splicing specific to different cell types have been identified in a few experimental systems (1,3,18,19,50,66), and progress in identifying transacting factors involved in tissue-specific regulation, particularly neuron-specific splicing, has been made. KH-type splicing regulatory protein is enriched in neurons and has been isolated as a component of a complex that activates inclusion of the c-src neuronal N1 exon (43). The KH-type RNA binding protein Nova-1 is expressed exclusively in neurons of the central nervous system (4) and activates inclusion of exons in the glycine receptor and GABA A receptor pre-mRNAs (27). Brain polypyrimidine tract binding protein (brPTB), a protein related to the more ubiquitous PTB but enriched in brain, binds to the same sequence recognized by PTB and antagonizes the effects of .In addition to the use of tissue-restricted factors, cell-specific alternative splicing may als...

Inclusion of cardiac troponin T (cTNT) exon 5 in embryonic muscle requires conserved flanking intronic elements (MSEs). ETR-3, a member of the CELF family, binds U/G motifs in two MSEs and directly activates exon inclusion in vitro. Binding and activation by ETR-3 are directly antagonized by polypyrimidine tract binding protein (PTB). We use dominant-negative mutants to demonstrate that endogenous CELF and PTB activities are required for MSE-dependent activation and repression in muscle and nonmuscle cells, respectively. Combined use of CELF and PTB dominant-negative mutants provides an in vivo demonstration that antagonistic splicing activities exist within the same cells. We conclude that cell-specific regulation results from the dominance of one among actively competing regulatory states rather than modulation of a nonregulated default state.

Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote musclespecific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66 -81 nucleotides downstream of exon 5, assembles a complex that is both sequenceand muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.Alternative splicing allows single genes to express multiple mRNAs. Splice site selection is often regulated in a cell-specific manner resulting in regulated expression of different protein isoforms (1-3). Genetic and biochemical studies in Drosophila have identified specific cis elements and trans-acting factors which mediate cell-specific splicing events (4). In vertebrates, cis elements that mediate cell-specific splicing events have been identified (5-12). Factors that bind to some of these elements have also been identified (13-18), but it remains unclear how these factors mediate cell-specific splicing events.We are using the chicken cardiac troponin T (cTNT) 1 gene to investigate the mechanisms of regulated splicing in striated muscle. cTNT expression is restricted to embryonic skeletal muscle and to embryonic and adult cardiac muscle. Exon 5 undergoes developmentally regulated splicing such that inclusion predominates in embryonic skeletal and cardiac muscle and skipping predominates in the adult (19). We have previously identified four cis-acting elements in the introns flanking exon 5 that function as muscle-specific splicing enhancers (MSEs) by transient transfection analysis of cTNT minigenes (5, 6). The MSEs are necessary for higher levels of exon inclusion in muscle cells than in fibroblast cells. Mutation of these elements causes exon skipping in muscle cells but has little effect on splicing in fibroblasts. These results have defined exon skipping as the default splicing pattern and indicate that exon inclusion requires positive-acting trans-factors present in muscle.The four MSEs are designated 1 to 4. MSE1 is located in intron 4, immediately upstream of exon 5. MSEs 2, 3, and 4 are located in the first 130 nucleotides of intron 5 (5). MSE3 includes nucleotides 66 -81 of intron 5 and was originally identified because of i...