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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 2012; 7(7):e40315Plos

Abstract

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/β-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.

Authors+Show Affiliations

Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del I.P.N., México DF, México.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22792276

Citation

Ortuño-Pineda, Carlos, et al. "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, vol. 7, no. 7, 2012, pp. e40315.
Ortuño-Pineda C, Galindo-Rosales JM, Calderón-Salinas JV, et al. 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. 2012;7(7):e40315.
Ortuño-Pineda, C., Galindo-Rosales, J. M., Calderón-Salinas, J. V., Villegas-Sepúlveda, N., Saucedo-Cárdenas, O., De Nova-Ocampo, M., & Valdés, J. (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), pp. e40315. doi:10.1371/journal.pone.0040315.
Ortuño-Pineda C, et al. 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. 2012;7(7):e40315. PubMed PMID: 22792276.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Ortuño-Pineda,Carlos, AU - Galindo-Rosales,José Manuel, AU - Calderón-Salinas,José Victor, AU - Villegas-Sepúlveda,Nicolás, AU - Saucedo-Cárdenas,Odila, AU - De Nova-Ocampo,Mónica, AU - Valdés,Jesús, Y1 - 2012/07/05/ PY - 2012/03/02/received PY - 2012/06/04/accepted PY - 2012/7/14/entrez PY - 2012/7/14/pubmed PY - 2013/4/3/medline SP - e40315 EP - e40315 JF - PloS one JO - PLoS ONE VL - 7 IS - 7 N2 - 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/β-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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/22792276/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_ L2 - http://dx.plos.org/10.1371/journal.pone.0040315 DB - PRIME DP - Unbound Medicine ER -