Figure 2. Binding of recombinant AtGRP8 to the AtGRP8 transcript. (A) GST-AtGRP8 protein was incubated with labelled 8-intron_WT ORN in the presence of 5 mg of tRNA and 2, 20, 100 and 250 pmol of unlabelled 8-intron_WT ORN (lanes 3–6) or 8-intron_G2U2mut (lanes 7-10), respectively. Lane 1, free ORN. (B) GST-AtGRP8 protein was incubated with labelled 8-UTR_WT ORN in the presence of 5 mg of tRNA and 2, 20, 100 and 250 pmol of unlabelled 8-UTR_WT ORN (lanes 3–6) or 8-UTR_G6mut (lanes 7-10), respectively. Lane 1, Nucleic Acids Research, 2008, Vol. 36, No. 22 6981 intron a strong interaction with GST-AtGRP8 was found which was abolished by 250 pmol of unlabeled 7-intron_WT but not aﬀected by 500 pmol of 7-intron G6mut (Figure 3B). This shows that AtGRP8 also binds to the AtGRP7 intron and 30 UTR with a certain speciﬁ- city. Thus, AtGRP7 and AtGRP8 may recognize overlap- ping or identical motifs within both pre-mRNAs. To compare the binding aﬃnities, Kd values were determined for all interactions (Table 2). For the four binding sites, the Kd values were in the same order of magnitude as those previously determined for GST-AtGRP7 protein (12). Therefore, presently we cannot infer which of the possible interactions between AtGRP7 or AtGRP8 and the respective target sites may prevail in vivo. Mutation of AtGRP8 RNP1 Arg47 impairs RNA-binding activity in vitro Binding of AtGRP8 to its own and the AtGRP7 pre- mRNA in vitro suggests that this interaction may initiate alternative splicing and down-regulation of endogenous AtGRP7 and AtGRP8 in vivo. To investigate whether this in vitro binding activity correlated with in vivo func- tion, a mutation was introduced into the RRM of recom- binant GST-AtGRP8. The conserved arginine (R) 47 predicted to lie at the beginning of the b3 strand that is part of the RNA-binding platform was exchanged for glu- tamine (Q) (25–27). Indeed, we have shown that recombi- nant GST-AtGRP7-RQ with an analogous mutation of R49 has a 6-fold lower aﬃnity for its target sites than WT GST-AtGRP7 protein while folding of the protein is not aﬀected (12). The AtGRP8-RQ mutant protein was expressed as GST-fusion and tested for binding to the AtGRP7 and AtGRP8 target sites. Only weak interaction was observed with Kd values about one order of magnitude higher for GST-AtGRP8-RQ compared to GST-AtGRP8 (Table 2). Competition assays revealed that 8-UTR_WT competed much better for binding of GST-AtGRP8-RQ than 8-UTR_G6mut and 7-UTR_WT competed much better for binding of GST-AtGRP8-RQ than 7-UTR_G4mut (data not shown). Thus, mutation of the conserved R47 reduces the binding aﬃnity rather than the speciﬁcity of the interaction. The AtGRP8 R47Q mutation impairs but does not abolish promotion of alternative AtGRP8 and AtGRP7 splicing To test the impact of the RQ mutation on AtGRP8 activ- ity in vivo, plants constitutively overexpressing the mutated AtGRP8-RQ protein were generated (AtGRP8- RQ-ox) and compared to plants overexpressing WT AtGRP8 protein (AtGRP8-ox). Transgenic plants were free ORN. (C) To compare binding aﬃnities, 50 fmol of labelled 8-UTR_WT ORN or 8-UTR_938 were incubated with 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2, 2.5, 5 and 10 mM or 0.1, 0.25, 0.5, 1, 2, 3, 4, 5 and 10 mM of GST-AtGRP8, respectively. All reactions contained 1 mg tRNA. Bound and free RNA were quantiﬁed and Kd values calculated based on the mean of three independent experiments as described (12). (D) GST-AtGRP8 protein was incubated with labelled 8-UTR_938 ORN in the presence of 5 mg of tRNA and 2, 20, 100 and 250 pmol of unlabelled 8-UTR_938 ORN (lanes 2–5) or 7-UTR_ G4mut (lanes 7–10), respectively. Lane 6, free ORN.