Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells
The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3’ss, three alternative 5’ss (N4-, N50-, and N62-5’ss) and a variety of putative target sites fo...
| Autores principales: | , , , , , , , |
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| Formato: | Artículo |
| Lenguaje: | inglés |
| Publicado: |
Public Library of Science
2012
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| Acceso en línea: | http://eprints.uanl.mx/15005/1/608.pdf |
| _version_ | 1824414221270515712 |
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| author | Buratti, Emanuele Ortuño Pineda, Carlos Galindo Rosales, José Manuel Calderón Salinas, José Victor Villegas Sepúlveda, Nicolás Saucedo Cárdenas, Odila De Nova Ocampo, Mónica Valdés, Jesús |
| author_facet | Buratti, Emanuele Ortuño Pineda, Carlos Galindo Rosales, José Manuel Calderón Salinas, José Victor Villegas Sepúlveda, Nicolás Saucedo Cárdenas, Odila De Nova Ocampo, Mónica Valdés, Jesús |
| author_sort | Buratti, Emanuele |
| collection | Repositorio Institucional |
| description | The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3’ss, three alternative 5’ss (N4-, N50-, and N62-5’ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5’ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5’ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/b-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5’ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5’ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes. |
| format | Article |
| id | eprints-15005 |
| institution | UANL |
| language | English |
| publishDate | 2012 |
| publisher | Public Library of Science |
| record_format | eprints |
| spelling | eprints-150052020-03-05T22:57:54Z http://eprints.uanl.mx/15005/ Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells Buratti, Emanuele Ortuño Pineda, Carlos Galindo Rosales, José Manuel Calderón Salinas, José Victor Villegas Sepúlveda, Nicolás Saucedo Cárdenas, Odila De Nova Ocampo, Mónica Valdés, Jesús The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3’ss, three alternative 5’ss (N4-, N50-, and N62-5’ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5’ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5’ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/b-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5’ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5’ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes. Public Library of Science 2012 Article PeerReviewed text en cc_by_nc_nd http://eprints.uanl.mx/15005/1/608.pdf http://eprints.uanl.mx/15005/1.haspreviewThumbnailVersion/608.pdf Buratti, Emanuele y Ortuño Pineda, Carlos y Galindo Rosales, José Manuel y Calderón Salinas, José Victor y Villegas Sepúlveda, Nicolás y Saucedo Cárdenas, Odila y De Nova Ocampo, Mónica y Valdés, Jesús (2012) Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells. PloS one, 7 (7). e40315. ISSN 1932-6203 doi:10.1371/journal.pone.0040315 |
| spellingShingle | Buratti, Emanuele Ortuño Pineda, Carlos Galindo Rosales, José Manuel Calderón Salinas, José Victor Villegas Sepúlveda, Nicolás Saucedo Cárdenas, Odila De Nova Ocampo, Mónica Valdés, Jesús Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells |
| thumbnail | https://rediab.uanl.mx/themes/sandal5/images/online.png |
| title | Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells |
| title_full | Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells |
| title_fullStr | Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells |
| title_full_unstemmed | Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells |
| title_short | Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells |
| title_sort | binding of hnrnp h and u2af65 to respective g codes and a poly uridine tract collaborate in the n50 5 ss selection of the rest n exon in h69 cells |
| url | http://eprints.uanl.mx/15005/1/608.pdf |
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