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. Author manuscript; available in PMC: 2016 May 7.
Published in final edited form as: Cell. 2015 May 7;161(4):762–773. doi: 10.1016/j.cell.2015.03.020

Figure 1. Switch RNA can bind AID.

Figure 1

(A) In vitro transcribed (IVT) telomeric and switch RNAs bind to AID. IVT biotinylated RNAs were folded and incubated with whole cell extracts from stimulated CH12 cells, followed by pull-down with streptavidin beads. Proteins recovered were analyzed by immunoblot with AID or Apobec3 antibodies; while bound RNAs were analyzed by dot blot using streptavidin-HRP. Input RNAs were verified to be biotinylated by streptavidin-HRP-northern blot. SμF, SαF; forward/sense switch μ and α RNA. SμR, SαR; reverse/anti-sense switch μ and α RNA. Result shown is representative of three independent pull-down experiments.

(B) Switch RNA interacts with AID in vivo. CH12 cells stably expressing S1-aptamer tagged Sα transcripts, in either the forward/sense (S1SαF) or the reverse/anti-sense (S1SαR) orientation, were stimulated. Untagged SαF and SαR expressing cells were used as controls. The S1-aptamer tag has affinity for streptavidin and ribonucleoprotein complexes were isolated on streptavidin beads. RNA in the eluates was reverse transcribed and analyzed by qPCR (RT-qPCR) for amounts of Sα transcripts relative to S1SαF, while proteins in the eluates were analyzed by immunoblot. Result shown is representative of three independent pull-down experiments.

(C) Competition RNA binding assay. RNA pull-down was performed with 1 nM biotinylated SμF RNA and 100 ng MBP-AID(WT) protein, in the presence of increasing concentrations of non-biotinylated competitor RNAs. Bound MBP-AID(WT) recovered by pull-down with streptavidin beads were analyzed by immunoblot with an AID antibody. Data shown is representative of three experiments.