Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16
Por:
Martínez-Salazar M., López-Urrutia E., Arechaga-Ocampo E., Bonilla-Moreno R., Martínez-Castillo M., Díaz-Hernández J., Del Moral-Hernández O., Cedilla-Barron, L, Martines-Juarez V., De Nova-Ocampo M., Valdes J., Berumen J., Villegas-Sepúlveda N.
Publicada:
5 dic 2014
Resumen:
The human papillomavirus type 16 (HPV-16) E6/E7 spliced transcripts are
heterogeneously expressed in cervical carcinoma. The heterogeneity of
the E6/E7 splicing profile might be in part due to the intrinsic
variation of splicing factors in tumor cells. However, the splicing
factors that bind the E6/E7 intron 1 (In-1) have not been defined.
Therefore, we aimed to identify these factors; we used HeLa nuclear
extracts (NE) for in vitro spliceosome assembly. The proteins were
allowed to bind to an RNA/DNA hybrid formed by the In-1 transcript and a
5'-biotinylated DNA oligonucleotide complementary to the upstream exon
sequence, which prevented interference in protein binding to the intron.
The hybrid probes bound with the nuclear proteins were coupled to
streptavidin magnetic beads for chromatography affinity purification.
Proteins were eluted and identified by mass spectrometry (MS).
Approximately 170 proteins were identified by MS, 80% of which were RNA
binding proteins, including canonical spliceosome core components,
helicases and regulatory splicing factors. The canonical factors were
identified as components of the spliceosomal B-complex. Although 35-40
of the identified factors were cognate splicing factors or helicases,
they have not been previously detected in spliceosome complexes that
were assembled using in vivo or in vitro models. (C) 2014 Elsevier B.V.
All rights reserved.
Filiaciones:
Martínez-Salazar M.:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo., Postal 14-740, México D.F., 07360, Mexico
Unidad de Investigación Médica en Inmunoquímica, Hospital de Especialidades del Centro Médico Nacional Siglo XXI IMSS, México D.F., 03020, Mexico
López-Urrutia E.:
Lab. Genómica Funcional UBIMED FES IZTACALA UNAM Estado de México, Mexico
Arechaga-Ocampo E.:
Departamento de Ciencias Naturales, División de Ciencias Naturales e Ingenieria, Universidad Autónoma Metropolitana-Cuajimalpa, Av. Vasco de Quiroga 4871, Col. Santa Fe Cuajimalpa de Morelos, D.F. C.P., 05300, Mexico
Bonilla-Moreno R.:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo., Postal 14-740, México D.F., 07360, Mexico
Martínez-Castillo M.:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo., Postal 14-740, México D.F., 07360, Mexico
Díaz-Hernández J.:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo., Postal 14-740, México D.F., 07360, Mexico
Del Moral-Hernández O.:
Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Ciudad Universitaria, Avenida Lázaro Cárdenas S/N, Chilpancingo, Gro, 39090, Mexico
Martines-Juarez V.:
Área Académica de Medicina Veterinaria y Zootecnia, Universidad Autónoma del estado de Hidalgo, Tulancingo, Hgo, Mexico
De Nova-Ocampo M.:
Programa Institucional de Biomedicina Molecular Escuela Nacional de Medicina y Homeopatía, IPN, México D.F., Mexico
Valdes J.:
Depto. Bioquímica, Centro de Investigación y de Estudios Avanzados-IPN (CINVESTAV-IPN), Unidad Zacatenco, México D.F., 07360, Mexico
Berumen J.:
Univ Nacl Autonoma Mexico, Fac Med, Mexico City 04510, DF, Mexico
Villegas-Sepúlveda N.:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo., Postal 14-740, México D.F., 07360, Mexico
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