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Nedeljković, Mirjana
(2012).
DOI: https://doi.org/10.21954/ou.ro.0000f0e4
Abstract
The beta adducin gene has a very complex architecture, with tissue-specific use of promoters and polyadenylation sites (PAS). Its expression is restricted to neuronal and hematopoietic tissues. In mice, usage of a distal brain-specific PAS (PAS4) generates an 8. 3 kb long mRNA with an unusually long 3’UTR of about 5.7 kb. Instead, the use of proximal erythroid-specific PASs results in short 3.1-3.7 kb mRNA isoforms. The presence and use of the different alternative PASs in the beta adducin gene are very well conserved among species suggesting an important and specific role for each of the generated 3’UTRs. To study the regulatory mechanisms involved in beta adducin polyadenylation, minigene constructs containing the mouse beta adducin polyadenylation signals were used. The constructs were transfected into HeLa cells and their expression and PAS selection determined by Northern blot analysis. The exclusive usage of the PAS4 in HeLa cells has been observed. All the elements defining the core polyadenylation signal were characterized: the hexanucleotide motif (Hm), the upstream and downstream sequence elements (USE and DSE, respectively) elements, and the cleavage site.
Interestingly, I have detected the presence of two novel non-canonical cis-acting elements regulating 3’end processing at the PAS4. Both elements act on long-distance and to the best of our knowledge, long-distance upstream polyadenylation regulatory elements have not previously been described for the non-viral eukaryotic transcripts. The first of these elements was essential to enable polyadenylation at the PAS4. It was located in a region spanning 355 nucleotides and includes the stop codon of beta adducin. The second non-canonical upstream polyadenylation regulatory element seems to inhibit processing at the PAS4. It was located in the region close to the second proximal PAS, about 4.5 kb upstream of the PAS4.
These results highlight the complexity of the regulatory mechanisms directing beta adducin pre-mRNA 3’end processing.
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