of evolutionarily related RNA sequences and an evolu- tionary tree relating them and predicts as output the con- served secondary structures supported by the evolutionary patterns detected in the input alignment. A set of nine pre- mRNA sequences comprising AtGRP8 orthologs with the same gene structure comprising a 50 UTR, two protein- coding exons of phase 0, a single intron and a 30 UTR (Table S1) were assembled. To generate the input align- ment, we ﬁrst compiled separate alignments for 50 UTRs, exon 1, intron, exon 2 and 30 UTRs using ClustalW (Version 1.83) (22), which we then combined manually into longer alignments. To maximize the alignment qual- ity, the alignments of the pre-mRNA sequences of exons 1 and 2 were based on the ClustalW alignments of the corresponding encoded amino-acid sequences. As the resulting full-length pre-mRNA alignment (1407 nt) was too long to analyze in a single chunk, we generated an mRNA input alignment with UTRs (1055 nt) to investi- gate conserved secondary structures in or near the UTRs and a pre-mRNA input alignment without UTRs (1093 nt) to investigate conserved secondary structures in or near the intron. RESULTS AND DISCUSSION AtGRP8 negatively autoregulates its own pre-mRNA To begin to investigate the molecular properties of the predicted RNA-binding protein AtGRP8, we generated transgenic plants ectopically overexpressing the AtGRP8 coding region under control of the CaMV promoter with the duplicated enhancer (AtGRP8-ox plants). Immuno- blot analysis using a speciﬁc AtGRP8 antibody identiﬁed transgenic lines with strongly elevated AtGRP8 protein levels (Figure 1A). Compared to WT plants, the total AtGRP8 transcript level was strongly elevated in AtGRP8-ox plants harvested at zt3 (zeitgeber time 3, that is 3 h after lights on), the circadian minimum of AtGRP8 oscillations, and zt11, the circadian maximum, due to the expression of the transgene (Figure 1C). The endogenous AtGRP8 transcript forms are selectively detected with a gene-speciﬁc probe derived from the 50 UTR that is not contained in the overexpression construct (Figure 1C). In WT plants, the endogenous AtGRP8 mRNA and a small amount of its pre-mRNA containing the 283-nt intron were present. In the AtGRP8-ox plants, almost no mature endogenous AtGRP8 mRNA was detectable. Instead, an intermediate size transcript appears at a low level, corresponding to the alternatively spliced AtGRP8 transcript (as_AtGRP8) that is generated through the use of a cryptic 50 splice site within the intron. These data indicate that AtGRP8 exerts negative autoregulation on its own pre-mRNA. In WT plants, as_AtGRP8 is hardly detectable. Nevertheless, corresponding cDNAs have been isolated from cDNA libraries (DS, unpublished results). In response to the elevated AtGRP8 protein level, a shift to a cryptic 50 splice site occurs and as_AtGRP8 accumulates to a low level at the expense of the mature mRNA in AtGRP8-ox plants. Because the retained part of the intron contains in-frame stop codons, no full-length Nucleic Acids Research, 2008, Vol. 36, No. 22 6979 Figure 1. Inﬂuence of ectopic AtGRP8 overexpression on the endogen- ous AtGRP7 and AtGRP8. (A) The immunoblot with total protein of WT plants, the AtGRP8-ox lines 5 and 21 and an AtGRP7-ox line, harvested at zt11, was probed with the AtGRP8 antibody (top) and an antibody against LHCP (light harvesting chlorophyll-binding protein) as loading control (bottom). The absence of crossreaction with over- expressed AtGRP7 protein in AtGRP7-ox plants demonstrates the spe- ciﬁcity of the antibody. (B) A duplicate blot was probed with the AtGRP7 antibody (top) and the LHCP antibody (bottom). (C) WT and AtGRP8-ox plants were harvested at zt3 and zt11. The RNA gel blot was hybridized with the AtGRP8 cDNA to determine the total AtGRP8 transcript level. The stripped blot was rehybridized with the gene-speciﬁc probe to monitor the endogenous AtGRP8 transcript and subsequently with the gene-speciﬁc AtGRP7 probe. The position of the pre-mRNA, as_AtGRP8 and as_AtGRP7 retaining the ﬁrst half of the intron and the mature mRNA are indicated. Boxes represent exons, lines represent the ﬁrst and second half of the intron, respectively. The ethidium-bromide stained gel shows equal loading. AtGRP8 protein can be produced from the as_AtGRP8 transcript. Thus, like AtGRP7, AtGRP8 negatively auto- regulates, and the molecular underpinnings of the AtGRP8 feedback loop are similar to the way AtGRP7 inﬂuences its own circadian oscillations (12,13). AtGRP8 promotes alternative splicing of the AtGRP7 pre-mRNA To test whether AtGRP8 would also inﬂuence AtGRP7 transcript abundance, the AtGRP7 transcript level was