UPDATED April 2008
Interactions involving Arabidopsis polyadenylation factor subunits
One approach towards understanding mRNA 3' end formation in plants is to characterize pairwise interactions involving plant polyadenylation factors. In this project, a number of approaches are being used to identify such interactions. These include a random combinatorial phage-display screen (Addepalli and Hunt, 2008), more standard two-hybrid assays (Hunt et al., 2008), in vitro pull-down assays using recombinant proteins expressed in E. coli (Elliott et al., 2003; Xu et al., 2006; Forbes et al., 2006; Xing et al., 2008), and co-immunoprecipitation assays using Arabidopsis nuclear extracts (Xu et al., 2006; Delaney et al., 2006; Xing et al., 2008).
This page summarizes the results of a large-scale compilation of pairwise interactions amongst polyadenylation factor subunits. This study has recently been published in BMC Genomics (Hunt et al., 2008), and experimental details may be found in this paper. In this study, each Arabidopsis polyadenylation factor subunit isoform (this includes the products of apparent duplicated genes) was tested for interactions with all other subunits using a standard yeast two-hybrid assay. Positive results were compiled and analyzed using Cytoscape, and the results tailored for display as shown in this page.
The following figure shows the summary of interactions that were observed in this study. The network has been displayed so as to illustrate the hypothetical complexes and hubs that are apparent. Several aspects of this network are interesting. Two probable complexes are readily apparent - these correspond to the so-called Cleavage and Polyadenylation Specificity Factor (or CPSF; these are the blue subunits in the figure) and the mammalian Cleavage Factor II (or CFIIm (light green subunits). In addition, two conceptual hubs may be discerned - these center on one of the Fip1 orthologs (FIPS5) and on CPSF30.
What does all of this mean? Presently, there are some interesting parallels with other eukaryotes and some intriguing differences. As is the case in other eukaryotes, the CPSF subunits appear to reside in a unified complex; this has been confirmed by co-purification assays performed with purified proteins and with nuclear extracts (Xu et al., 2006; Delaney et al., 2006; Herr et al., 2006). There are differences in the behaviors of the four PAP (black subunits) and three PabN (gray subunits) isoforms; whether these will be reflected in other assays remains to be determined, but the possibilitity that the various isoforms have undergone functional specialization is tantalizing. The hubs that center around FIPS5 and CPSF30 are densely-populated, perhaps moreso than is consistent with the existence of a static complex in which all of the displayed interactions are in force. An alternative model that is being explored holds the observed interactions reflect two phenomena - a progression through the 3' processing reaction, such that various subunits are rearranged, with some subunits replacing others in a corresponding position in the complex, and/or the existence of different complexes that act on subsets of polyadenylation signals.
Figure legend: Summary of the set of protein-protein interactions revealed by the two-hybrid assays (Hunt et al., 2008). Interactions were compiled and displayed using the Cytoscape software package.
Addepalli B, Hunt AG. 2008. The interaction between two Arabidopsis polyadenylation factor subunits involves an evolutionarily-conserved motif and has implications for the assembly and function of the polyadenylation complex. Protein Peptide Letters 15: 76-88. (request a reprint)
Delaney KJ, Xu R, Zhang J, Yun K-Y, Li QQ, Falcone DF, Hunt AG. 2006. Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiol. 140: 1507-1521 (request a reprint)
Elliott BJ, Dattaroy T, Meeks-Midkiff LR, Forbes KP, Hunt AG. 2003. An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDa subunit of CPSF. Plant Mol Biol 51:373-84. (request a reprint)
Forbes KP, Addepalli B, Hunt AG. 2006. An Arabidopsis Fip1 homologue interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits. J. Biol. Chem. 281: 176-186. (request a reprint)
Herr AJ, Molnar A, Jones A, Baulcombe DC. 2006. Defective RNA processing enhances RNA silencing and influences flowering of Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 14994-14501.
Hunt AG, Xu R, Addepalli B, Rao S, Forbes KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhya A, Dampanaboina L, Marion A, Von Lanken C, Li QQ. 2008. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling. BMC Genomics 9, 220. (Open Access article)
Xu R, Zhao H, Dinkins RD, Cheng X, Carberry G, Li QQ. 2006. The 73 kD subunit of the Cleavage and Polyadenylation Specificity Factor (CPSF) Complex affects reproductive development in Arabidopsis. Plant Mol Biol 61, 799-815. (request a reprint)
Xing D, Zhao H, Xu R, Li QQ. 2008. Arabidopsis PCFS4, a homologue of yeast polyadenylation factor Pcf11p, regulates FCA alternative processing and promotes flowering time. Plant J, in press. (request a reprint)