6982 Nucleic Acids Research, 2008, Vol. 36, No. 22 Figure 3. Binding of recombinant AtGRP8 to the AtGRP7 transcript. (A) GST-AtGRP8 protein was incubated with labelled 7-UTR_WT ORN in the presence of 5 mg of tRNA and 2, 20, 100 and 250 pmol of unlabelled 7-UTR_WT ORN (lanes 3–6) or 20, 100, 250 and 500 pmol of unlabelled 7-UTR_G4mut (lanes 7–10), respectively. Lane 1, free ORN. (B) GST-AtGRP8 protein was incubated with labelled 7-intron_WT ORN in the presence of 5 mg of tRNA and 2, 20, 100 and 250 pmol of unlabelled 7-intron_WT ORN (lanes 3–6) or 20, 100, 250 and 500 pmol of unlabelled 7-intron_G6mut ORN (lanes 7–10), respectively. Lane 1, free ORN. identiﬁed with a total AtGRP8 transcript level constitu- tively elevated at zt3 and zt11 (Figure 4A, top). An immu- noblot analysis conﬁrmed expression of intact mutated protein, as total AtGRP8 protein was elevated to a level similar to that in AtGRP8-ox plants overexpressing WT AtGRP8 protein (Figure 4B). However, in contrast to AtGRP8-ox plants, the mature endogenous AtGRP8 mRNA was still detected in addition to as_AtGRP8 (Figure 4A, middle). Thus, an elevated AtGRP8-RQ level only partially shifts the splice site in favor of the cryptic 50 splice site with concomitant reduction in as_AtGRP8 abundance, as observed in AtGRP8-ox plants. We infer from this that the weakened interaction of AtGRP8-RQ with its own pre-mRNA impairs but does not abolish negative autoregulation of the AtGRP8 pre-mRNA. Overexpression of WT AtGRP8 led to an accumulation of as_AtGRP7 and an almost complete loss of the mature mRNA (Figure 4C). In contrast, no alternatively spliced AtGRP7 transcript was detected in AtGRP8-RQ-ox plants. Thus, the R47Q mutation reduces the eﬀect AtGRP8 protein has on splice site selection. The level of mature AtGRP7 mRNA was only weakly reduced Table 2. Kd values for recombinant GST-AtGRP8-RQ and GST- AtGRP8 ORN Protein Kd [M] Kd RQ/Kd WT 8-UTR_WT GST-AtGRP8-WT 2.05 GST-AtGRP8-RQ 1.31 7-UTR_WT GST-AtGRP8-WT 3.18 GST-AtGRP8-RQ 3.22 8-intron_WT GST-AtGRP8-WT 1.64 GST-AtGRP8-RQ 2.29 7-intron_WT GST-AtGRP8-WT 4.26 GST-AtGRP8-RQ 3.59 0.03 0.35 2.27 0.97 0.65 0.36 0.52 2.02 10–7 10–6 6.4 10–7 10–6 10.1 10–6 10–5 14.0 10–7 10–6 8.4 Kd values were determined as described in (12). The ratio between the Kd value for the mutated protein and the WT protein is indicated. compared to WT plants, consistent with the idea that the residual binding activity of AtGRP8-RQ still causes some production of as_AtGRP7 at the expense of the AtGRP7 mRNA. However, the alternatively spliced tran- script is hardly detectable due to its short half life (13). Accordingly, almost no AtGRP7 protein was detected in AtGRP8-ox plants, whereas a small amount of AtGRP7 protein was detectable in AtGRP8-RQ-ox lines (Figure 4D). Altogether, the interaction of AtGRP8 with the AtGRP7 and AtGRP8 pre-mRNAs in vivo is relevant for the negative autoregulation and crossregulation. Presumably, binding of AtGRP8 and AtGRP7 triggers additional factors that act in concert to regulate the choice of splice sites (12). Distinct cold response of AtGRP8 and AtGRP7 The reciprocal regulation between AtGRP7 and AtGRP8 uncovered here may suggest that the proteins are able to fully substitute for each other. Therefore we compared their expression patterns. According to publically avail- able microarray data, AtGRP7 and AtGRP8 are expressed at a similar level across plant tissues except for a lower AtGRP8 abundance in siliques and later stages of seed development (Figure S2) (28). Moreover, the circadian maximum of AtGRP8 transcript oscillation is only slightly advanced relative to the AtGRP7 peak (29). Both AtGRP7 and AtGRP8 transcripts have been described as being upregulated by cold (11,30). We investigated steady-state protein levels under these conditions using speciﬁc anti- peptide antibodies. Two-week-old Col plants were trans- ferred to 48C at light onset and harvested without cold treatment and after 1, 2, 4 and 7 days, respectively. AtGRP8 protein abundance rose marginally at best after transfer to 48C, whereas AtGRP7 protein abundance stea- dily increased up to day 7 (Figure 5A, B). The AtGRP8 transcript level was only weakly increased at the circadian minimum (zt2) after 1 day of cold treatment but declined afterwards (Figure 5C). Also at the circadian maximum (zt10), AtGRP8 transcript levels declined during extended exposure to cold. In contrast, a strong increase of the AtGRP7 transcript level was observed at the circadian minimum (zt2). At the circadian maximum (zt10), the level in plants transferred to 48C was not elevated beyond that of control plants. These data indicate that AtGRP7 and AtGRP8 diﬀer in their response to cold