Final Activity Report Summary - FCA/FY_COMPLEX (Definition of the protein components of the FCA/FY complex in Arabidopsis thaliana)
We have undertaken an extensive search for FY interacting proteins by using different techniques like TAP tag purification and yeast two hybrid screenings. As a result of this work, we are currently writing a paper addressing the characterisation of the FY complex and reporting an unexpected mechanism of regulation of FY protein levels.
FY interacts with FCA to limit the accumulation of FLC, a MADS-box transcription factor that is a potent repressor of flowering in Arabidopsis thaliana. FCA / FY interaction is also required for the alternative polyadenylation of FCA mRNA itself, conferring a feedback mechanism that controls the levels of functional FCA gamma transcript. FY interacts with FCA through sequences within the PPLP motifs present in its C-terminal region. The seven WD-repeats in the N-terminal half of the FY protein suggest that additional proteins interact with FY to form a larger multi-subunit complex. FY is a homologue of PFS2P, a yeast polyadenylation factor that promotes the assembly of the RNA processing complexes CPII-PF1 and CF1A through its interaction with Ysh1p, Fip1p and RNA14p. In order to elucidate the function FY does in vivo and to explore the extent of similarity between the yeast mRNA polyadenylation complex and the Arabidopsis FCA / FY complex, we have undertaken a search for FY interacting proteins. Yeast two hybrid experiments show that FY cannot interact with CPSF-73 and CstF-77 (Arabidopsis proteins, homogolous to yeast Ysh1p and RNA14p respectively). Moreover, FY cannot complement pfs2p mutations in yeast. These results suggest that there are differences in the architecture of mRNA processing / polyadenylation complexes of plants and yeast. To clarify this question, we generated transgenic plants carrying an FYTAP construct where FY cDNA was fused to the sequence of TAP tag under the control of FY promoter. Analysis of transformed plants showed that FYTAP chimeric protein mostly complemented fy-2 mutation. Interestingly, FYTAP protein was accumulated at notably higher levels than FY endogenous protein in wt Col plants, suggesting the existence of a regulatory mechanism controlling the levels of FY protein. TAP tag purification from seedlings show that FY interacts with at least two subunits of the Cleavage and specificity polyadenylation factor(s), CPSF160 and CPS100, thus confirming our previous negative results in yeast and showing that FY interacts with some other components of the polyadenylation complex. However, no FCA peptides were found to be in the FYTAP sample, suggesting that FY and FCA might be interacting transiently in vivo. To address this question, we analysed FY and FCA complexes by size exclusion chromatography. Results show that two different FY complexes exist in vivo, and that the largest one is disrupted in a fy-2 background (an allele where PLPP motifs are lost and therefore the resulting truncated protein is unable to interact with FCA). This result suggests that FY performs essential polyadenylation functions separately from specific functions, such as flowering time regulation.
We searched for more FY interacting proteins by yeast two hybrid screening using FY (WD repeats) and a library made from Col FRI seedlings. We found three interacting clones corresponding to At3g12570. This gene encodes for a highly conserved plant specific expressed protein with a predicted Zn carboxypeptidase domain at its N-terminus and a heat shock protein 20-like domain at its C-terminus. Characterisation of the corresponding SALK insertion line shows that FY protein levels are upregulated in the mutant, whereas FY mRNA levels are not, suggesting the existence of a post-transcriptional mechanism for the regulation of FY protein. Interestingly when we expressed FY cDNA under the control of 35S promoter in Arabidopsis plants, we did not find any changes of FY protein levels, even though there was a massive accumulation of FY mRNA. On the contrary, FYTAP tag plants showed exceedingly higher levels of FYTAP protein but no changes of FYTAP mRNA levels. All these observations confirm the existence of a post-transcriptional regulatory mechanism that regulates FY protein levels, most likely acting through the action of a protease-like activity.
FY interacts with FCA to limit the accumulation of FLC, a MADS-box transcription factor that is a potent repressor of flowering in Arabidopsis thaliana. FCA / FY interaction is also required for the alternative polyadenylation of FCA mRNA itself, conferring a feedback mechanism that controls the levels of functional FCA gamma transcript. FY interacts with FCA through sequences within the PPLP motifs present in its C-terminal region. The seven WD-repeats in the N-terminal half of the FY protein suggest that additional proteins interact with FY to form a larger multi-subunit complex. FY is a homologue of PFS2P, a yeast polyadenylation factor that promotes the assembly of the RNA processing complexes CPII-PF1 and CF1A through its interaction with Ysh1p, Fip1p and RNA14p. In order to elucidate the function FY does in vivo and to explore the extent of similarity between the yeast mRNA polyadenylation complex and the Arabidopsis FCA / FY complex, we have undertaken a search for FY interacting proteins. Yeast two hybrid experiments show that FY cannot interact with CPSF-73 and CstF-77 (Arabidopsis proteins, homogolous to yeast Ysh1p and RNA14p respectively). Moreover, FY cannot complement pfs2p mutations in yeast. These results suggest that there are differences in the architecture of mRNA processing / polyadenylation complexes of plants and yeast. To clarify this question, we generated transgenic plants carrying an FYTAP construct where FY cDNA was fused to the sequence of TAP tag under the control of FY promoter. Analysis of transformed plants showed that FYTAP chimeric protein mostly complemented fy-2 mutation. Interestingly, FYTAP protein was accumulated at notably higher levels than FY endogenous protein in wt Col plants, suggesting the existence of a regulatory mechanism controlling the levels of FY protein. TAP tag purification from seedlings show that FY interacts with at least two subunits of the Cleavage and specificity polyadenylation factor(s), CPSF160 and CPS100, thus confirming our previous negative results in yeast and showing that FY interacts with some other components of the polyadenylation complex. However, no FCA peptides were found to be in the FYTAP sample, suggesting that FY and FCA might be interacting transiently in vivo. To address this question, we analysed FY and FCA complexes by size exclusion chromatography. Results show that two different FY complexes exist in vivo, and that the largest one is disrupted in a fy-2 background (an allele where PLPP motifs are lost and therefore the resulting truncated protein is unable to interact with FCA). This result suggests that FY performs essential polyadenylation functions separately from specific functions, such as flowering time regulation.
We searched for more FY interacting proteins by yeast two hybrid screening using FY (WD repeats) and a library made from Col FRI seedlings. We found three interacting clones corresponding to At3g12570. This gene encodes for a highly conserved plant specific expressed protein with a predicted Zn carboxypeptidase domain at its N-terminus and a heat shock protein 20-like domain at its C-terminus. Characterisation of the corresponding SALK insertion line shows that FY protein levels are upregulated in the mutant, whereas FY mRNA levels are not, suggesting the existence of a post-transcriptional mechanism for the regulation of FY protein. Interestingly when we expressed FY cDNA under the control of 35S promoter in Arabidopsis plants, we did not find any changes of FY protein levels, even though there was a massive accumulation of FY mRNA. On the contrary, FYTAP tag plants showed exceedingly higher levels of FYTAP protein but no changes of FYTAP mRNA levels. All these observations confirm the existence of a post-transcriptional regulatory mechanism that regulates FY protein levels, most likely acting through the action of a protease-like activity.