6980 Nucleic Acids Research, 2008, Vol. 36, No. 22 Table 1. Sequences of oligoribonucloetides used as RNA-binding substrates Name Sequence 8-UTR_WT GUUUUUGGUUUAGAUUUGGUUUUGUGU 8-UTR_G6mut GUUUUUAAUUUAAAUUUAAUUUUAUGU 8-UTR_938 CGUUUGGUUUACUUUUUUGAUGAAACA 8-intron_WT CUUCCACGAUUGUUUUUGCUGAUGUGU 8-intron_G2U2mut CUUCCACGAUUAUCCUUACUGAUGUGU 7-UTR_WT AUUUUGUUCUGGUUCUGCUUUAGAUUUGAUCU 7-UTR_G4mut AUUUUAUUCUAAUUCUGCUUUAGAUUUAAUCU 7-intron_WT GUUCAGUUUUGUUGGAUUGUUUUGCUGAUCUG 7-intron_G6mut GUUCAAUUUUAUUAAAUUAUUUUACUGAUCUG Sequences of the AtGRP8 30 UTR (8-UTR_WT and 8-UTR_938) and intron (8-intron_WT) ORN and the corresponding mutated 8-UTR_G6mut and 8-intron_G2U2mut with six G residues exchanged for A or two G and two U residues exchanged for A or C (in bold), respectively, as well as sequence of AtGRP7 30 UTR (7-UTR_WT) and intron (7-intron_WT) ORN and the corresponding mutated 7-UTR_G4mut and 7-intron_G6mut with four or six G residues exchanged for A (in bold), respectively, are shown. compared in WT and AtGRP8-ox plants (Figure 1C). In WT plants, the mature AtGRP7 mRNA and a small amount of the pre-mRNA were detected at the circadian maximum. When AtGRP8 was expressed at high levels, a low amount of the as_AtGRP7 transcript appeared at the expense of the mature mRNA. Accordingly, AtGRP7 pro- tein was barely detectable in the AtGRP8-ox plants (Figure 1B). AtGRP8 RNA and protein steady-state abundance pre- viously have been shown to be under negative control by AtGRP7 (12,13). Consistent with this, AtGRP8 RNA and protein levels are elevated in the atgrp7-1 T-DNA inser- tion mutant due to relief of repression (15). Our data now show that not only does AtGRP7 inﬂuence AtGRP8 abun- dance, but also AtGRP8 in turn regulates AtGRP7. Similarly, another Arabidopsis splicing regulator, the Ser/Arg-rich (SR) protein atRSZ33 crossregulates alterna- tive splicing of a conserved long intron in the atRSp31 gene, equivalent to the eﬀect on its own pre-mRNA (23,24). The alternative atRSp31 form accumulates to high levels and thus is stable in contrast to as_AtGRP8. Recombinant AtGRP8 binds to its own pre-mRNA As AtGRP7 autoregulation involves AtGRP7 binding to its own 30 UTR and intron, it is conceivable that AtGRP8 might similarly interact with its own pre-mRNA. Therefore, we performed RNA-bandshift assays to test the AtGRP7-binding sites determined previously (12) for interaction with recombinant AtGRP8 (Table 1). For the intronic binding site 8-intron_WT GST-AtGRP8 formed a retarded complex which was competed more eﬃciently by the ORN 8-intron_WT than by the mutated counterpart 8-intron_G2U2mut with exchanges of two G and two U residues (Figure 2A). For the 30 UTR, a retarded complex was observed for 8-UTR_WT that was more eﬃciently outcompeted by unlabeled ORN 8-UTR_WT than by the mutated counterpart 8-UTR_G6mut with mutation of six G residues (Figure 2B). Sequence align- ment of the AtGRP7-binding sites upon the AtGRP8 intron and 30 UTR had uncovered a second motif with homology to the AtGRP7-binding site within the AtGRP8 30 UTR (data not shown). However, determina- tion of the Kd values revealed that AtGRP8 has a 10-fold lower aﬃnity for the corresponding ORN 8-UTR_938 (Table 1) compared to 8-UTR_WT (Figure 2C). Further- more, the complex was outcompeted by an excess of unla- beled 8-UTR_938 to the same degree as by a negative control ORN 7-UTR_G4mut (Table 1) (Figure 2D). Based on the low aﬃnity and lack of speciﬁcity, 8-UTR_938 was excluded as binding site. Taken together, our data show that AtGRP8 binds to its own pre-mRNA and recognizes sequences in the second half of the intron and the 30 UTR that also comprise targets for AtGRP7. To assess the secondary structure of these putative bind- ing sites in the context of the entire pre-mRNA, AtGRP8 was searched for secondary structure elements conserved in plant orthologs with RNA-Decoder (20,21). Among the numerous existing secondary structure prediction pro- grams, RNA-Decoder is unique in that it explicitly takes the known protein-coding regions of an input alignment into account. This feature is important when searching partly protein-coding sequences such as pre-mRNAs for conserved secondary structure elements, as the evolution- ary pattern due to amino-acid conservation needs to be carefully distinguished from the evolutionary pattern due to secondary structure conservation (20,21). In order to detect conserved secondary structures in the AtGRP8 pre-mRNA, we assembled a set of nine pre- mRNA sequences comprising AtGRP8 as well as eight AtGRP8 orthologs from the Brassicaceae mustard and oilseed rape, the other dicotyledoneous plants tobacco and Pelargonium, and the monocotyledoneous plants rice and maize (Table S1). As input tree to RNA- Decoder, we used the tree predicted by ClustalW (22) for the nine encoded protein sequences. The predicted secondary structures for the AtGRP8 and, for compari- son, the AtGRP7 pre-mRNAs are shown in Figure S1. Interestingly, the predicted AtGRP8-binding sequences map to conserved regions, pointing to an important role of the secondary structure for the function of these motifs. 8-intron_WT and 8-UTR_WT are predicted to be single- stranded. In contrast, 8-UTR_938 is predicted to be par- tially double-stranded and thus may be less accessible to interacting proteins, in line with the very low binding aﬃ- nity (Figure 2C). Recombinant AtGRP8 binds to the AtGRP7 pre-mRNA The data shown in Figure 1 indicate that AtGRP8 nega- tively inﬂuences AtGRP7 pre-mRNA splicing in the same way as AtGRP7 autoregulates. As the shift to the alter- native splice form relies on AtGRP7 binding to its own pre-mRNA, we tested whether recombinant AtGRP8 would bind in vitro to the AtGRP7 target sites within the AtGRP7 pre-mRNA (Table 1). Complex formation was observed with 7-UTR_WT that was completely lost upon addition of 250 pmol of unlabeled 7-UTR_WT, but much less reduced with 500 pmol of the mutated 7-UTR_G4mut (Figure 3A). Also for the ORN spanning the AtGRP7-binding site within the second half of the