Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit.
The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.
Delaney
Kimberly J
KJ
Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546-0312, USA.
Xu
Ruqiang
R
Zhang
Jingxian
J
Li
Q Quinn
QQ
Yun
Kil-Young
KY
Falcone
Deane L
DL
Hunt
Arthur G
AG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D039223:Cleavage And Polyadenylation Specificity Factor / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D016335:Zinc Fingers
D029681:Arabidopsis Proteins; D002147:Calmodulin; D039223:Cleavage And Polyadenylation Specificity Factor; D021122:Protein Subunits