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Alternative polyadenylation in human COX‐2: trans‐acting factors
Author(s) -
Lutz Carol S.,
HallPogar Tyra,
Liang Songchun,
Hague Lisa K.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a282
Subject(s) - polyadenylation , downregulation and upregulation , rna , cleavage and polyadenylation specificity factor , microbiology and biotechnology , messenger rna , gene , biology , chemistry , genetics
Poly(A) tails are found on most mammalian mRNAs. A “canonical” polyadenylation signal has been defined as an upstream AAUAAA hexamer core element plus a U/UG downstream core element. The reality of polyadenylation signals found in human genes, however, is quite different. Therefore, alternative polyadenylation is an important yet relatively unexplored mechanism of gene regulation. We have focused on alternative polyadenylation of human cyclooxygenase‐2 (COX‐2), which is regulated at many levels. While COX‐1 is constitutively expressed, COX‐2 is inducible and is upregulated in response to many signals. Since increased transcriptional activity accounts for only part of the upregulation of COX‐2, we chose to explore other RNA processing mechanisms in the regulation of this gene. We have previously shown that COX‐2 is regulated by alternative polyadenylation, that the COX‐2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs), and that these USEs are very important in efficient polyadenylation (Hall‐Pogar et al., 2005). Our previous results suggest that trans‐acting factors interacting with COX‐2 USEs are very important in COX‐2 polyadenylation. To explore what trans‐acting factors may be involved, we performed a pull‐down assay with HeLa nuclear extract and biotinylated RNA oligonucleotides representing a COX‐2 USE. Specifically bound proteins were identified by mass spectrometry and were verified by Western blotting. We identified PSF, p54nrb, PTB and U1A as proteins which specifically bound to the COX‐2 USEs. We have further explored their participation in polyadenylation using MS2 coat protein tethering assays.