Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16

Martha Martínez-Salazar, Eduardo López-Urrutia, Elena Arechaga-Ocampo, Raul Bonilla-Moreno, Macario Martínez-Castillo, Job Díaz-Hernández, Oscar Del Moral-Hernández, Leticia Cedillo-Barrón, Víctor Martines-Juarez, Monica De Nova-Ocampo, Jesús Valdes, Jaime Berumen, Nicolás Villegas-Sepúlveda

Research output: Contribution to journalArticle

Abstract

© 2014 Elsevier B.V. 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. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: César López-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.
Original languageAmerican English
Pages (from-to)184-197
Number of pages164
JournalJournal of Proteomics
DOIs
StatePublished - 5 Dec 2014

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Spliceosomes
Human papillomavirus 16
Proteomics
Introns
Statistical Factor Analysis
Mass spectrometry
Mass Spectrometry
Affinity chromatography
Proteins
RNA-Binding Proteins
Streptavidin
DNA
Nuclear Proteins
Oligonucleotides
Purification
Victoria
Tumors
Exons
Affinity Chromatography
Cells

Cite this

Martínez-Salazar, M., López-Urrutia, E., Arechaga-Ocampo, E., Bonilla-Moreno, R., Martínez-Castillo, M., Díaz-Hernández, J., ... Villegas-Sepúlveda, N. (2014). Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16. Journal of Proteomics, 184-197. https://doi.org/10.1016/j.jprot.2014.07.029
Martínez-Salazar, Martha ; López-Urrutia, Eduardo ; Arechaga-Ocampo, Elena ; Bonilla-Moreno, Raul ; Martínez-Castillo, Macario ; Díaz-Hernández, Job ; Del Moral-Hernández, Oscar ; Cedillo-Barrón, Leticia ; Martines-Juarez, Víctor ; De Nova-Ocampo, Monica ; Valdes, Jesús ; Berumen, Jaime ; Villegas-Sepúlveda, Nicolás. / Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16. In: Journal of Proteomics. 2014 ; pp. 184-197.
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Martínez-Salazar, M, López-Urrutia, E, Arechaga-Ocampo, E, Bonilla-Moreno, R, Martínez-Castillo, M, Díaz-Hernández, J, Del Moral-Hernández, O, Cedillo-Barrón, L, Martines-Juarez, V, De Nova-Ocampo, M, Valdes, J, Berumen, J & Villegas-Sepúlveda, N 2014, 'Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16', Journal of Proteomics, pp. 184-197. https://doi.org/10.1016/j.jprot.2014.07.029

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16. / Martínez-Salazar, Martha; López-Urrutia, Eduardo; Arechaga-Ocampo, Elena; Bonilla-Moreno, Raul; Martínez-Castillo, Macario; Díaz-Hernández, Job; Del Moral-Hernández, Oscar; Cedillo-Barrón, Leticia; Martines-Juarez, Víctor; De Nova-Ocampo, Monica; Valdes, Jesús; Berumen, Jaime; Villegas-Sepúlveda, Nicolás.

In: Journal of Proteomics, 05.12.2014, p. 184-197.

Research output: Contribution to journalArticle

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T1 - Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16

AU - Martínez-Salazar, Martha

AU - López-Urrutia, Eduardo

AU - Arechaga-Ocampo, Elena

AU - Bonilla-Moreno, Raul

AU - Martínez-Castillo, Macario

AU - Díaz-Hernández, Job

AU - Del Moral-Hernández, Oscar

AU - Cedillo-Barrón, Leticia

AU - Martines-Juarez, Víctor

AU - De Nova-Ocampo, Monica

AU - Valdes, Jesús

AU - Berumen, Jaime

AU - Villegas-Sepúlveda, Nicolás

PY - 2014/12/5

Y1 - 2014/12/5

N2 - © 2014 Elsevier B.V. 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. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: César López-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.

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Martínez-Salazar M, López-Urrutia E, Arechaga-Ocampo E, Bonilla-Moreno R, Martínez-Castillo M, Díaz-Hernández J et al. Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16. Journal of Proteomics. 2014 Dec 5;184-197. https://doi.org/10.1016/j.jprot.2014.07.029