University of Kentucky
RNA Binding by the 30 kDa Subunit of Cleavage and Polyadenylation Stimulation Factor of Arabidopsis thaliana
Institution
University of Kentucky
Faculty Advisor/ Mentor
Arthur Hunt
Abstract
The Arabidopsis thaliana homolog of mammalian Cleavage and Polyadenylation Specificity Factor, 30 kDa (CPSF 30) is an active part of the Arabidopsis polyadenylation complex. In plants there are three conserved polyadenylation signals: 1) the cleavage sites which is the point where precursor RNA is cleaved and adenosine residues are added, 2) the NUE (near upstream element, 6-10 base pairs long, situated 10-40 bases 5’ of the cleavage site), an A-rich signaling element thought to possibly function similarly to the highly conserved AAUAAA sequence in mammals, and 3) the FUE (far upstream element, that can be as large at 100 nucleotides and lie anywhere from 13 to 100 nucleotides 5’ of the NUE), a U-rich region with multiple UG motifs. CPSF 30 showed binding of relatively equal strength to wild type RNA (containing all polyadenylation signals) and RNA with no effective NUE; however, CPSF 30 displays decreased binding to RNA containing no active signals. This suggests CPSF 30 preferentially binds the FUE region of mRNA. RNA binding was also examined in the presence of polynucleotide competitors. CPSF 30 showed little to no binding to wild type RNA in the presence of Poly G and Poly U and reduced binding in the presence of Poly A, indicating the protein has an affinity for these poly neucleotides over any of the polyadenylation signals. The preference for Poly G and Poly U further supports the hypothesis that CPSF 30 binds at the FUE region of mRNA.
RNA Binding by the 30 kDa Subunit of Cleavage and Polyadenylation Stimulation Factor of Arabidopsis thaliana
The Arabidopsis thaliana homolog of mammalian Cleavage and Polyadenylation Specificity Factor, 30 kDa (CPSF 30) is an active part of the Arabidopsis polyadenylation complex. In plants there are three conserved polyadenylation signals: 1) the cleavage sites which is the point where precursor RNA is cleaved and adenosine residues are added, 2) the NUE (near upstream element, 6-10 base pairs long, situated 10-40 bases 5’ of the cleavage site), an A-rich signaling element thought to possibly function similarly to the highly conserved AAUAAA sequence in mammals, and 3) the FUE (far upstream element, that can be as large at 100 nucleotides and lie anywhere from 13 to 100 nucleotides 5’ of the NUE), a U-rich region with multiple UG motifs. CPSF 30 showed binding of relatively equal strength to wild type RNA (containing all polyadenylation signals) and RNA with no effective NUE; however, CPSF 30 displays decreased binding to RNA containing no active signals. This suggests CPSF 30 preferentially binds the FUE region of mRNA. RNA binding was also examined in the presence of polynucleotide competitors. CPSF 30 showed little to no binding to wild type RNA in the presence of Poly G and Poly U and reduced binding in the presence of Poly A, indicating the protein has an affinity for these poly neucleotides over any of the polyadenylation signals. The preference for Poly G and Poly U further supports the hypothesis that CPSF 30 binds at the FUE region of mRNA.