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Enhancing polyadenylation in vivo with bioinformatically identified auxiliary cis ‐elements
Author(s) -
Darmon Sarah K.,
Tian Bin,
Lutz Carol S.
Publication year - 2009
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.23.1_supplement.662.3
Subject(s) - polyadenylation , upstream (networking) , cleavage and polyadenylation specificity factor , untranslated region , three prime untranslated region , random hexamer , messenger rna , biology , microbiology and biotechnology , genetics , chemistry , gene , computer science , telecommunications
Polyadenylation is a processing event eukaryotic cells use to make mature translatable mRNA. Cis ‐elements in the 3' untranslated region (UTR) of mRNA aid in efficiency of polyadenylation. Upstream of the polyadenylation site (PAS) is a hexamer that is most often AAUAAA. Downstream is a U/GU‐rich sequence. Both elements bind core polyadenylation factors. Additional auxiliary cis ‐elements further upstream or downstream of the PAS were previously identified in a bioinformatic survey. We used this predicted information to direct our in vivo validation studies. Auxiliary elements were placed in their respective sites in the cyclooxygenase‐2 (COX‐2) 3'UTR. An in vivo polyadenylation assay determined the strength of the polyadenylation signal. Upstream elements (USEs) enhanced usage of the COX‐2 PAS three to four fold. Downstream elements (DSEs) enhanced usage two fold. A luciferase assay showed increased protein levels with the insertion of the auxiliary elements. USEs had greater enhancement in protein levels compared to DSEs. Overall, some auxiliary elements had more enhancing effects than others, with some elements giving little effect as compared to wild type. We conclude that auxiliary elements have a role in enhancing polyadenylation and may aid in polyadenylation choice during alternative polyadenylation. Research supported by NIH and the Foundation of UMDNJ grants.