title: The intron 1 of HPV 16 has a suboptimal branch point at a guanosine.
abstract: The branch point sequence (BPS) of intron 1 of the HPV-16 was determined via RT-PCR in a cell free system, using lariat intermediates obtained by in vitro splicing reactions. We used synthetic E6/E7 transcripts and HeLa nuclear protein extracts to obtain the splicing intermediates. Then, a divergent oligonucleotide primer set, pairing on the lariat RNA that encompassed the 2'-5' phosphodiester bond formed between the 5' end of the intron and the BPS, was used for cDNA synthesis and PCR amplification. Subsequent RT-PCR assays revealed four splicing intermediates, made up of a major intermediary corresponding to the BPS and four cryptic branched sequences. Only intermediates bound at the 5' end of the intron are probably the authentic branch point sequence, and all of them branch at guanosine 328 instead of the typical adenosine. Unusually, the BPS of intron 1 of HPV-16 is a suboptimal sequence (AGUGAGU) that differs from the eukaryotic consensus BPS, which correlates with the splicing profile observed for early transcripts of HPV-16 in tumors and tumor derived cell lines. The implications of this unusual branch point sequence for splicing of the HPV-16 pre-mRNA are discussed.
authors.affiliation: Depto. Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados, México.
authors.forename: Marco Antonio
authors.initials: MA
authors.lastname: De la Rosa-Rios
authors.forename: Martha
authors.initials: M
authors.lastname: Martínez-Salazar
authors.forename: Martha
authors.initials: M
authors.lastname: Martínez-Garcia
authors.forename: César
authors.initials: C
authors.lastname: González-Bonilla
authors.forename: Nicolás
authors.initials: N
authors.lastname: Villegas-Sepúlveda
mesh_terms: D002457:Cell Extracts; D006151:Guanosine / Q000235:genetics*; D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D007438:Introns; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D002457:Cell Extracts; D012333:RNA, Messenger; D012367:RNA, Viral; D006151:Guanosine