The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis

Abstract

Cytosine DNA methylation is an epigenetic mark frequently associated with silencing of genes and transposons. In Arabidopsis, the establishment of cytosine DNA methylation is performed by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). DRM2 is guided to target sequences by small interfering RNAs (siRNAs) in a pathway termed RNA-directed DNA methylation (RdDM). We performed a screen for mutants that affect the establishment of DNA methylation by investigating genes that contain predicted RNA-interacting domains. After transforming FWA into 429 T-DNA insertion lines, we assayed for mutants that exhibited a late-flowering phenotype due to hypomethylated, thus ectopically expressed, copies of FWA. A T-DNA insertion line within the coding region of the spliceosome gene SR45 (sr45-1) flowered late after FWA transformation. Additionally, sr45-1 mutants display defects in the maintenance of DNA methylation. DNA methylation establishment and maintenance defects present in sr45-1 mutants are enhanced in dcl3-1 mutant background, suggesting a synergistic cooperation between SR45 and DICER-LIKE3 (DCL3) in the RdDM pathway.

Keywords: :

• Arabidopsis
• de novo
• DNA Methylation
• genetic screen
• siRNA

Acknowledgments

We would like to thank Craig Pikaard for kindly donating anti-AGO4 immunosera and the expression vector for the peptide used for purification. We would like to thank members of the S. Jacobsen laboratory for supportive discussions. Jacobsen lab research was supported by US National Institutes of Health grant GM60398. I.A. was supported by a postdoctoral fellowship from the Ministerio de Educacion y Ciencia. M.V.C.G. was supported by USPHS National Research Service Award GM07104. C.F.L. was supported by the Ruth L. Kirschstein National Research Service Award GM07185.