title: Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16.
abstract: 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.
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo. Postal 14-740, 07360, México D.F., Mexico; Unidad de Investigación Médica en Inmunoquímica, Hospital de Especialidades del Centro Médico Nacional "Siglo XXI" IMSS, 03020 México D.F., Mexico.
authors.forename: Martha
authors.initials: M
authors.lastname: Martínez-Salazar
authors.affiliation: Lab. Genómica Funcional UBIMED FES IZTACALA UNAM Estado de México, Mexico.
authors.forename: Eduardo
authors.initials: E
authors.lastname: López-Urrutia
authors.affiliation: 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.
authors.forename: Elena
authors.initials: E
authors.lastname: Arechaga-Ocampo
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo. Postal 14-740, 07360, México D.F., Mexico.
authors.forename: Raul
authors.initials: R
authors.lastname: Bonilla-Moreno
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo. Postal 14-740, 07360, México D.F., Mexico.
authors.forename: Macario
authors.initials: M
authors.lastname: Martínez-Castillo
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo. Postal 14-740, 07360, México D.F., Mexico.
authors.forename: Job
authors.initials: J
authors.lastname: Díaz-Hernández
authors.affiliation: Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Avenida Lázaro Cárdenas S/N, Ciudad Universitaria, 39090 Chilpancingo, Gro, Mexico.
authors.forename: Oscar
authors.initials: O
authors.lastname: Del Moral-Hernández
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo. Postal 14-740, 07360, México D.F., Mexico.
authors.forename: Leticia
authors.initials: L
authors.lastname: Cedillo-Barrón
authors.affiliation: Área Académica de Medicina Veterinaria y Zootecnia, Universidad Autónoma del estado de Hidalgo, Tulancingo, Hgo, Mexico.
authors.forename: Víctor
authors.initials: V
authors.lastname: Martines-Juarez
authors.affiliation: Programa Institucional de Biomedicina Molecular Escuela Nacional de Medicina y Homeopatía, IPN, México D.F., Mexico.
authors.forename: Monica
authors.initials: M
authors.lastname: De Nova-Ocampo
authors.affiliation: Depto. Bioquímica, Centro de Investigación y de Estudios Avanzados-IPN (CINVESTAV-IPN), Unidad Zacatenco, 07360 México D.F., Mexico.
authors.forename: Jesús
authors.initials: J
authors.lastname: Valdes
authors.affiliation: Facultad de Medicina, UNAM, 04510 México D.F., Mexico; Unidad de Medicina Genómica, Hospital General, México D.F., Mexico.
authors.forename: Jaime
authors.initials: J
authors.lastname: Berumen
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) Apdo. Postal 14-740, 07360, México D.F., Mexico. Electronic address: nvillega@cinvestav.mx.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Villegas-Sepúlveda
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D004265:DNA Helicases / Q000378:metabolism; D005091:Exons; D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000737:chemistry*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry; D050725:Papillomavirus E7 Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry; D017381:Spliceosomes / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D020543:Proteome; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D004265:DNA Helicases
keywords: Alternative splicing; E6/E7(pre-mRNA); HPV-16 intron-1; Proteomics; Spliceosomal factors