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 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

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Resumen

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.

Tipo de elemento: Article
Divisiones: Medicina
Usuario depositante: Lic. Josimar Pulido
Creadores:
CreadorEmailORCID
Buratti, EmanueleNO ESPECIFICADONO ESPECIFICADO
Ortuño Pineda, CarlosNO ESPECIFICADONO ESPECIFICADO
Galindo Rosales, José ManuelNO ESPECIFICADONO ESPECIFICADO
Calderón Salinas, José VictorNO ESPECIFICADONO ESPECIFICADO
Villegas Sepúlveda, NicolásNO ESPECIFICADONO ESPECIFICADO
Saucedo Cárdenas, OdilaNO ESPECIFICADONO ESPECIFICADO
De Nova Ocampo, MónicaNO ESPECIFICADONO ESPECIFICADO
Valdés, JesúsNO ESPECIFICADONO ESPECIFICADO
Fecha del depósito: 09 Mayo 2019 22:18
Última modificación: 09 Mayo 2019 22:18
URI: http://eprints.uanl.mx/id/eprint/15005

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