Figure 1.

IDN2 Interacts with lncRNA and SWI3B

(A and B) IDN2 interacts with Pol V-produced lncRNA. RNA immunoprecipitation was performed with anti-IDN2 antibody in Col-0 wild-type, nrpe1 mutant, and idn2-1 mutant with a deletion in the RNA-binding XS domain. Recovered RNA was digested with DNase I and assayed by real-time RT-PCR (A) or reverse transcribed and amplified followed by real-time PCR (B). ACTIN2 signal serves as a loading control. Graphs show averages normalized to the wild-type and SD from four (A) or two (B) biological repeats. Input, no antibody and no RT controls as well as RNA IP results normalized to wild-type inputs are shown in Figure S1A.

(C) Domain structure of SWI3B and SWI3B clones identified with the yeast two-hybrid screen with IDN2 as bait.

(D) IDN2 interacts with SWI3B but not with its homologs, SWI3A, SWI3C, or SWI3D. Interaction of full-length SWI3A, SWI3B, SWI3C, and SWI3D with IDN2 was tested with a yeast two-hybrid assay. A series of three 10× dilutions is shown. Yeast growth on a plate with His is shown as a loading control.

(E) IDN2 interacts with SWI3B in tobacco. GFP-tagged SWI3B was coexpressed in tobacco leaves with FLAG-tagged IDN2. After immunoprecipitation with anti-GFP antibody the samples were analyzed by western blot with anti-FLAG antibody. Plants expressing only single construct were used as controls. Total protein extracts (inputs) were assayed by western blot to demonstrate comparable protein expression levels. The asterisk indicates a nonspecific band. Reciprocal coimmunoprecipitation is shown in Figure S1C.

(F) IDN2 interacts with SWI3B in Arabidopsis. GFP-tagged SWI3B and FLAG-tagged IDN2 under the control of their respective native promoters were transformed into Arabidopsis. Obtained transgenic lines were crossed and analyzed by coimmunoprecipitation with anti-GFP antibody and western blot with anti-FLAG antibody. Asterisks indicate nonspecific bands.

See also Figure S1.