RESUMO
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.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Galinhas , Éxons/genética , Humanos , Íntrons/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Troponina T/genética , Troponina T/metabolismoRESUMO
Members of the CUG-BP and ETR-3 like factor (CELF) protein family bind within conserved intronic elements (called MSEs) flanking the cardiac troponin T (cTNT) alternative exon 5 and promote exon inclusion in vivo and in vitro. Here we use a comparative deletion analysis of two family members (ETR-3 and CELF4) to identify separate domains required for RNA binding and splicing activity in vivo. CELF proteins contain two adjacent RNA binding domains (RRM1 and RRM2) near the N-terminus and one RRM (RRM3) near the C-terminus, which are separated by a 160-230 residue divergent domain of unknown function. Either RRM1 or RRM2 of CELF4 are necessary and sufficient for binding MSE RNA and RRM2 plus an additional 66 amino acids of the divergent domain are as effective as full-length protein in activating MSE-dependent splicing in vivo. Non-overlapping N- and C-terminal regions of ETR-3 containing either RRM1 and RRM2 or RRM3 plus segments of the adjacent divergent domain activate MSE-dependent exon inclusion demonstrating an unusual functional redundancy of the N- and C-termini of the protein. These results identify specific regions of ETR-3 and CELF4 that are likely targets of protein-protein interactions required for splicing activation.
Assuntos
Splicing de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Proteínas CELF , Células Cultivadas , Embrião de Galinha , Humanos , Íntrons , Proteínas do Tecido Nervoso , Estrutura Terciária de Proteína , Codorniz , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Deleção de SequênciaRESUMO
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.
Assuntos
Processamento Alternativo/genética , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Sequência de Bases , Western Blotting , Humanos , Íntrons , Dados de Sequência Molecular , Músculo Esquelético/patologia , Distrofia Miotônica/classificação , Distrofia Miotônica/patologia , Especificidade de Órgãos , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
