title: Characterization of the distal polyadenylation site of the ß-adducin (Add2) pre-mRNA.
abstract: Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Costessi
authors.forename: Fabiola
authors.initials: F
authors.lastname: Porro
authors.forename: Alessandra
authors.initials: A
authors.lastname: Iaconcig
authors.forename: Mirjana
authors.initials: M
authors.lastname: Nedeljkovic
authors.forename: Andrés Fernando
authors.initials: AF
authors.lastname: Muro
mesh_terms: D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D023061:Gene Order; D006801:Humans; D009154:Mutation; D059372:Nucleotide Motifs; D011061:Poly A; D026723:Polyadenylation; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D000099291:Adducins
chemical_list: D002148:Calmodulin-Binding Proteins; D011061:Poly A; D012322:RNA Precursors; D015534:Trans-Activators; D000099291:Adducins
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