RESUMO
Alternative splicing of exon 6 in Fas pre-mRNA generates a membrane bound pro-apoptotic isoform or soluble anti-apoptotic isoform. SRSF4 is a member of Arginine-Serine rich (SR) protein family. Here we demonstrate that increased SRSF4 expression stimulates exon 6 inclusion, and that reduced SRSF4 expression promotes exon 6 exclusion. We also show that weaker but not stronger 5' splice-site strength of exon 6 abolishes the SRSF4 effects on exon 6 splicing. Furthermore, we identified a novel enhancer on exon 6, on which SRSF4 interacts functionally and physically. Our results illustrate a novel regulatory mechanism of Fas pre-mRNA splicing.
Assuntos
Elementos Facilitadores Genéticos/genética , Éxons/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Receptor fas/genética , Sequência de Bases , Regulação da Expressão Gênica , Células HCT116 , Humanos , Ligação Proteica/genética , Precursores de RNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor fas/metabolismoRESUMO
U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF(65) effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF(65) even inhibits general splicing of adenovirus major late (Ad ML) or ß-globin pre-mRNA. Thus, we conclude that U2AF(65) possesses a splicing Inhibitory function that leads to alternative exon skipping.
Assuntos
Processamento Alternativo/genética , Éxons/genética , Proteínas Nucleares/genética , Ribonucleoproteínas/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Íntrons/genética , Dados de Sequência Molecular , Proteínas Nucleares/química , Ligação Proteica , Estrutura Terciária de Proteína , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Ribonucleoproteínas/química , Proteínas do Complexo SMN/genética , Fator de Processamento U2AF , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Proteínas Virais/genética , Globinas beta/genética , Proteínas tau/genéticaRESUMO
The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3' splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3' splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.
Assuntos
Íntrons/genética , Proteínas Nucleares/metabolismo , Sítios de Splice de RNA/genética , Splicing de RNA , RNA Nuclear Pequeno/genética , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Immunoblotting , Dados de Sequência Molecular , Proteínas Nucleares/genética , Filogenia , Interferência de RNA , Ribonucleoproteínas/genética , Spliceossomos/metabolismo , Fator de Processamento U2AFRESUMO
CD44 is a complex cell adhesion molecule that mediates communication and adhesion between adjacent cells as well as between cells and the extracellular matrix. CD44 pre-mRNA produces various mRNA isoforms through alternative splicing of 20 exons, among which exons 1-5 (C1-C5) and 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. CD44 V10 exon has important roles in breast tumor progression and Hodgkin lymphoma. Here we show that increased expression of hnRNP L inhibits V10 exon splicing of CD44 pre-mRNA, whereas reduced expression of hnRNP L promotes V10 exon splicing. In addition, hnRNP L also promotes V10 splicing of endogenous CD44 pre-mRNA. Through mutation analysis, we demonstrate that the effects of hnRNP L on V10 splicing are abolished when the CA-rich sequence on the upstream intron of V10 exon is disrupted. However, hnRNP L effects are stronger if more CA-repeats are provided. Furthermore, we show that hnRNP L directly contacts the CA-rich sequence. Importantly, we provide evidences that hnRNP L inhibits U2AF65 binding on the upstream Py tract of V10 exon. Our results reveal that hnRNP L is a new regulator for CD44 V10 exon splicing.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo L/biossíntese , Receptores de Hialuronatos/genética , Íntrons/genética , Splicing de RNA/genética , Adesão Celular/genética , Éxons/genética , Regulação da Expressão Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
The mouse immunoglobulin (IgM) pre-mRNA contains a splicing inhibitor that bears multiple binding sites for the splicing repressor polypyrimidine tract binding protein (PTB). Here we show that the inhibitor directs assembly of an ATP-dependent complex that contains PTB and U1 and U2 small nuclear RNAs (snRNAs). Unexpectedly, although U2 snRNA is present in the inhibitor complex, it is not base-paired to the branch point. We present evidence that inhibitor-bound PTB contacts U2 snRNA to promote base-pairing to an adjacent branch point-like sequence within the inhibitor, thereby preventing the U2 snRNA-branch point interaction and resulting in splicing repression. Our studies reveal a novel mechanism by which PTB represses splicing.
Assuntos
Pareamento de Bases/genética , Imunoglobulina M/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Precursores de RNA/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Trifosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Reagentes de Ligações Cruzadas/farmacologia , Éxons/genética , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Spliceossomos/genéticaRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of the spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA, fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNP protein family, when knocked down, promotes exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. We also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2ß. We present evidence that hnRNP M and tra2ß contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA.
Assuntos
Elementos Facilitadores Genéticos/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Atrofia Muscular Espinal/genética , Células do Corno Anterior/metabolismo , Células do Corno Anterior/patologia , Técnicas de Cultura de Células , Éxons/genética , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Terapia de Alvo Molecular , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/etiologia , Atrofia Muscular Espinal/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina , Medula Espinal/metabolismo , Medula Espinal/patologia , Fator de Processamento U2AF , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations of SMN1 cause SMA, a gene that encodes a SMN protein. SMN is important for the assembly of Sm proteins onto UsnRNA to UsnRNP. SMN has also been suggested to direct axonal transport of ß-actin mRNA in neurons. Humans contain a second SMN gene called SMN2 thus SMA patients produce some SMN but not with sufficient levels. The majority of SMN2 mRNA does not include exon 7. Here we show that increased expression of PSF promotes inclusion of exon 7 in the SMN2 whereas reduced expression of PSF promotes exon 7 skipping. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2ß in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.
Assuntos
Éxons/genética , Regulação Neoplásica da Expressão Gênica , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Western Blotting , Primers do DNA/química , Primers do DNA/genética , Humanos , Luciferases/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fator de Processamento Associado a PTB , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Células Tumorais CultivadasRESUMO
Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.
Assuntos
Processamento Alternativo/genética , Apoptose/genética , Caspase 2/genética , Proteínas de Ligação a RNA/genética , Sítios de Ligação/genética , Caspase 2/biossíntese , Éxons/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Fatores de Processamento de Serina-ArgininaRESUMO
The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ronâ³165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.
Assuntos
Proteínas Nucleares/fisiologia , Splicing de RNA , Receptores Proteína Tirosina Quinases/genética , Ribonucleoproteínas/fisiologia , Transcrição Gênica , Células Cultivadas , Éxons/genética , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proto-Oncogene Mas , Proto-Oncogenes/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
Spinal muscular atrophy (SMA) is caused by deletion of SMN1, one of the two SMN (Survival of Motor Neuron) genes. SMN2 is still present in SMA patients but its exon 7 is alternatively spliced. In this study, we took advantage of recent deposit of deep-sequencing datasets from a number of human tissues and examined the expression and splicing of SMN mRNA. We showed that SMN is slightly more abundantly expressed in the lungs and prostate. More interestingly, we found that the percentage of SMNΔ7 in lung and adipose tissue is significantly higher than in other tissues such as the testes, where almost no SMNΔ7 is expressed. Since SMN1 produces more than 95 % of full-length SMN RNA, it is likely that SMN2 has a higher skipping rate of exon 7 in adipose and lung tissue, leading to a higher ratio of SMNΔ7/SMN in these tissues. Given that many SMA patients die from respiratory failure, we speculate that higher skipping of exon 7 in the lungs may play a role in the progression of SMA.