FY Is an RNA 3 End - Processing Factor that Interacts with FCA to Control the Arabidopsis Floral

and controls the expression of a floral repressor, FLOWERING LOCUS C (FLC) (Michaels and Amasino, 1999; Sheldon et al., 1999), which modulates the activity of the other two pathways (Mouradov et al., 2002; Simpson and Dean, 2002). Gordon G. Simpson,1,2 Paul P. Dijkwel,1,3 Victor Quesada,4 Ian Henderson, and Caroline Dean* Department of Cell and Developmental Biology John Innes Centre The genetically defined autonomous pathway curNorwich rently comprises six mutants, fca, fy, fpa, fve, ld, and United Kingdom fld (Simpson and Dean, 2002). FCA and FPA encode RNA binding proteins (Macknight et al., 1997; Schomburg et al., 2001), and LD encodes a homeodomain proSummary tein (Lee et al., 1994), while FVE, FY, and FLD have not yet been characterized. We have been studying FCA The nuclear RNA binding protein, FCA, promotes Arabin order to understand the role and regulation of the idopsis reproductive development. FCA contains a autonomous pathway in flowering time control. FCA is WW protein interaction domain that is essential for a nuclear protein with two RNA recognition motif (RRM)FCA function. We have identified FY as a protein parttype RNA binding domains that can bind RNA in vitro ner for this domain. FY belongs to a highly conserved (Macknight et al., 1997; Quesada et al., 2003). In addition, group of eukaryotic proteins represented in SaccharoFCA contains a WW domain. This conserved protein myces cerevisiae by the RNA 3 end-processing factor, interaction module is found in a range of eukaryotic Pfs2p. FY regulates RNA 3 end processing in Arabiproteins that function in diverse cellular processes (Sudopsis as evidenced through its role in FCA regulation. dol and Hunter, 2000). WW domains have recently been FCA expression is autoregulated through the use of classified into four groups on the basis of their ligand different polyadenylation sites within the FCA prespecificity (Bedford et al., 2000; Sudol and Hunter, 2000). mRNA, and the FCA/FY interaction is required for effiThe resolution of cocrystal structures of WW domains cient selection of the promoter-proximal polyadenylacomplexed with their ligands has led to a clearer undertion site. The FCA/FY interaction is also required for standing of the mechanism by which specificity in these the downregulation of the floral repressor FLC. We interactions is determined (Bedford et al., 2000; Huang propose that FCA controls 3 end formation of specific et al., 2000; Verdecia et al., 2000; Zarrinpar and Lim, transcripts and that in higher eukaryotes, proteins ho2000). This classification therefore provides a predictive mologous to FY may have evolved as sites of associatool for identifying WW domain ligands. tion for regulators of RNA 3 end processing. The regulation of FCA expression is complex. FCA pre-mRNA is alternatively processed, resulting in the Introduction formation of four different transcripts, , , , and (Macknight et al., 1997). We have discovered that a prinThe floral transition is a major developmental switch in cipal level of control involves negative feedback of exflowering plants. Arabidopsis flowering time is regupression mediated by FCA itself (Quesada et al., 2003). lated by the quantitative integration of environmental Full-length FCA protein (FCA) promotes premature signals with an endogenous program of development cleavage and polyadenylation at a promoter-proximal (Mouradov et al., 2002; Simpson and Dean, 2002). Genes site within intron 3 of its own pre-mRNA. This results in required for the promotion of flowering have been identithe production of a nonfunctional truncated transcript, fied through the characterization of late-flowering muknown as FCA, at the expense of the fully spliced tants that comprise genetically separable pathways transcript, FCA, which encodes the active protein. The (Mouradov et al., 2002; Simpson and Dean, 2002). Mutaautoregulation of FCA expression is developmentally tions that delay flowering in longbut not short-day regulated and has a functional consequence for flowphotoperiods have been classified in the photoperiod ering time control (Quesada et al., 2003). FCA autoregupromotion pathway. Mutations affecting biosynthesis lation presets the level of active FCA expression (which of, or response to, the phytohormone gibberellin have is typically limiting; Macknight et al., 2002), which in turn a minor effect on flowering in long-day photoperiods controls the level of FLC mRNA. When the negative but can drastically delay flowering in short-day photoperegulation of FCA expression is bypassed, the balance riods. A third pathway, the autonomous pathway, proof the pathways controlling FLC is perturbed and flowmotes flowering in longand short-day photoperiods ering time is accelerated (Quesada et al., 2003). In order to determine the mechanism by which FCA controls the floral transition, we searched for proteins *Correspondence: [email protected] These authors contributed equally to this work. that interacted with FCA through its WW domain. We Present address: Gene Expression Programme, Scottish Crop Rehave found that FCA interacts with FY, a previously search Institute, Invergowrie, Dundee DD2 5DA, Scotland. uncharacterized component of the autonomous pathPresent address: Molecular Biology of Plants, University of Gronway. FY encodes a protein that is highly conserved in ingen, Kerklaan 30, 9751 NN Haren, The Netherlands. eukaryotes, and its homolog in S. cerevisiae functions Present address: Division de Genetica, Departamento de Biologia in pre-mRNA 3 end formation. We show that FY is reAplicada, Universidad Miguel Hernandez, Campus de Elche, Edificio