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 journalArticlepeer-review

2 Scopus citations

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. 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 languageEnglish
Pages (from-to)184-197
Number of pages14
JournalJournal of Proteomics
Volume111
DOIs
StatePublished - 5 Dec 2014

Keywords

  • Alternative splicing
  • E6/E7
  • HPV-16 intron-1
  • Proteomics
  • Spliceosomal factors

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