Fig. 2

Validation of VcAGO3 and small RNA properties. a β-elimination experiment performed with total RNA from gonidia (reproductive cells) as well as from somatic cells showing that small RNAs in V. carteri are modified and do not shift upon periodate treatment. Control siRNA carries an unprotected 3′OH and therefore shifts towards a smaller size. b Total RNA from somatic cells and reproductive cells in different life stages (vegetative growth, V; induced to the female differentiation program, I; fully differentiated to female, F) was used to validate miRNA by RNA blot. U6 serves as a loading control. All blots shown originate from one membrane. See methods for details on stripping and re-probing of the membrane. c Cleavage assay with VcAGO3, Arabidopsis thaliana (Ath) AGO1 (positive control) and CrGFP (negative control). For VcAGO3, three different small RNAs were selected according to read abundance in the VcAGO3 library, for AtAGO1, two known and abundant miRNAs were chosen. Immunoprecipitated Ago proteins (loaded with their endogenously bound small RNAs) were incubated with 5′ radiolabeled target RNA carrying a perfectly complementary site to the respective small RNA. After incubation, RNA was extracted and run on a gel. To indicate the position of the cleavage product, each target RNA was digested with RNase T1 (cleaves after G’s) to serve as a ladder. The bar on the side of the gel indicates the site of the complementary sequence and putative cleavage site. d Western blot showing the successful immunoprecipitation of myc-VcAGO3, myc-CrGFP and Flag-AthAGO1 used for the cleavage experiment in (c). Western blot was performed with part of the immunoprecipitation reaction used for the cleavage assay. Black bars on the side indicate the location of the bands