The autonomous promotion pathway plays a major role in controlling the floral transition in Arabidopsis. Its main function is to down-regulate RNA levels of the floral repressor, FLC. The Dean lab has cloned two components of this pathway, FCA and FY, which encode an RNA-binding protein and a homologue of a yeast protein required for mRNA 3'-end-processing and polyadenylation respectively. They have recently shown that FCA/FY proteins interact physically and regulate the alternative processing of the FCA tr anscript. Since FCA has no homology to any known component of the core polyadenylation machinery, it might be a regulator of 3'-end formation, whereas FY seems to be a conserved component of the 3'-end processing machinery.
The characterization of Arabidop sis FCA/FY complex will help to elucidate general questions about the composition of complexes regulating RNA 3'-end formation in eukaryotes. We expect to define the components of the FCA/FY complex by means of multidisciplinary proteomic approaches. The FY yeast homologue (Pfs2p) interacts with Yshlp, Fiplp, and RNA14p. We shall first clone Arabidopsis orthologues of Yshlp, Fiplp, and RNA14p, and we will test if the FY protein interacts with them by GST pull-down assay. Secondly, we will also identify other components of the FCA/FY complex by yeast two-hybrid experiments using FY as a bait.
Positive clones will be cloned into His-tagged vectors for efficient in vitro transcription/translation and co-immunoprecipitation experiments will be carried out with GS T-tagged FY protein. Finally,we will express FCA and FY-tagged proteins with TAP and HA epitopes in cultured Arabidopsis cells (both under the control of 35S and native promoters) and in Arabidopsis transgenic plants (under the control of native promoters) for the purification of the FCA/FY complex by affinity chromatography. We will identify these components by MudPIT. This methodology is now well established in Dean's lab.
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