Poster abstracts

Poster number 10 submitted by Lavanya Dampanaboina

Characterization of FY, an orthologue of yeast 3’ end processing factor Pfs2p, in plant polyadenylation using tethering assay and 3’ RACE.

Lavanya Dampanaboina (Plant and Soil Sciences, University of Kentucky), Dr. Arthur G Hunt (Plant and Soil Sciences, University of Kentucky)

Abstract:
Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA from all RNA polII transcripts in eukaryotes. Three cis-elements - FUE (Far upstream element), NUE (near upstream element) and cleavage site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes CPSF (cleavage and polyadenyation specificity factor), CstF (cleavage stimulation factor) along with cleavage factors and poly(A) polymerase (Zhao, Hyman et al. 1999). FY is a WD repeat protein, a 3’ end processing factor and is one of the subunits of the plant polyadenylation machinery. It is an ortholog of yeast essential processing factor Pfs2p that is involved in assembling different polyadenylation factors in cleavage and polyadenylation process (Ohnacker, Barabino et al. 2000). Yeast Pfs2p null mutants are lethal while Arabidopsis FY null alleles are embryo lethal and deleterious to growth in Nicotiana (Henderson, Liu et al. 2005).

FY is a part of the autonomous pathway, one of the flowering pathways regulating the transition from vegetative phase to the reproductive phase in Arabidopsis. FY targets FLC for repression by associating with another nuclear RNA binding protein, FCA. The FY-FCA complex also auto-regulates FCA expression by the selection of proximal polyadenylation site resulting in a truncated FCA protein (Henderson, Liu et al. 2005). Recent reports also show that FY regulates the polyadenylation site choice of FLC (Feng, Jacob et al. 2011). Here we employed a tethering assay to test the functional role of FY in 3’ end processing. To accomplish this MS2 coat protein is fused with FY and MS2 binding sites are added to the 3’UTR of the GFP reporter. To visualize the functional assay, transient studies were done using Agroinfiltrations in N.benthamiana. Further 3’RACE is used to confirm the visual results from transient studies. Results in these lines will be presented.

References:
1. Feng, W., Y. Jacob, et al. (2011).

Keywords: 3end processing, Tethering assay, RACE