Poster abstracts
Poster number 84 submitted by Braden Burdick
Using TurboID to Identify FY-Associated Proteins in Arabidopsis thaliana
Braden Burdick (Department of Plant and Soil Sciences, University of Kentucky), Lichun Zhou (Department of Plant and Soil Sciences, University of Kentucky), Carol D. Von Lanken (Department of Plant and Soil Sciences, University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)
Abstract:
In eukaryotic organisms, polyadenylation is a key step in messenger RNA processing that regulates gene expression. In Arabidopsis thaliana, the polyadenylation complex is made up of a number of interacting proteins that allow this process to occur, including the proteins FY and CPSF30 (Cleavage and Polyadenylation Specificity Factor 30). FY and CPSF30 work closely together to recognize the poly(A) signal [1] and recruit other polyadenylation proteins for cleavage and addition of a poly(A) tail at a specific site.
CPSF30 is an essential protein in most eukaryotes. However, it is dispensible in plants, as plants that do not express CPSF30 are viable [2]. This suggests that, in plants, CPSF30 can be removed or replaced within the polyadenylation complex, and perhaps that inhibition of its activity plays roles in the regulation of alternative polyadenylation. We hypothesize that, in the absence of CPSF30, another protein takes its place in the polyadenylation complex, allowing the plant to continue to process and polyadenylate mRNAs. In order to test this, we tagged the FY protein with TurboID, an enzyme that performs labeling of nearby proteins with biotin, and expressed the protein wild-type Col-0 and CPSF30-deficient plants (oxt6). FY was selected because of the close proximity of the mammalian ortholog (WDR33) to CPSF30 in the mammalian polyadenylation complex [3, 4], a proximity that was confirmed by structural modeling using AlphaFold. Immunoblot experiments showed that FY-TurboID was successfully expressed in wild-type Arabidopsis. Analysis of biotinylated proteins in transgenic lines reveals the existence of several novel proteins whose sizes are not consistent with those of the two CPSF30 isoforms. These results support our hypothesis by revealing several proteins (apart from CPSF30) that lie in close proximity to FY. We are analyzing biotinylated proteins in oxt6 plants that express FY-TurboID to confirm this result. Future experiments will include proteomic analysis of purified biotinylated proteins to determine which proteins may compensate for the loss of CPSF30 function.
References:
1.Yu, Z.B., J.C. Lin, and Q.S.Q. Li, Transcriptome Analyses of FY Mutants Reveal Its Role in mRNA Alternative Polyadenylation. Plant Cell, 2019. 31(10): p. 23322352.
2.Delaney, K.J., et al., Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiol, 2006. 140(4): p. 150721.
3.Chan, S.L., et al., CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3 ' processing. Genes & Development, 2014. 28(21): p. 23702380.
4.Schonemann, L., et al., Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33. Genes & Development, 2014. 28(21): p. 23812393.
Keywords: mRNA polyadenylation, CPSF30, TurboID