6978 Nucleic Acids Research, 2008, Vol. 36, No. 22 AtGRP8 oscillations are strongly depressed (9). This reg- ulation occurs at the post-transcriptional level through reduction of constitutive splicing and stimulation of alter- native splicing at a cryptic intronic 50 splice site of the AtGRP8 pre-mRNA, leading to an alternative splice var- iant (as_AtGRP8) with a premature termination codon (PTC) in the retained part of the intron. AtGRP7 uses the same mechanism for negative autore- gulation: rising protein levels promote alternative splicing by binding to its own pre-mRNA, and the alternatively spliced transcript (as_AtGRP7) is degraded via a pathway involving the nonsense-mediated decay (NMD) compo- nents AtUPF1 (UP FRAMESHIFT PROTEIN 1) and AtUPF3 (12,13). Thus, AtGRP7 is part of a negative feed- back loop through which it inﬂuences its own oscillation at the post-transcriptional level. This feedback loop is thought to operate as a slave oscillator downstream of the circadian clock, transducing temporal information within the cell by regulating other transcripts (13,14). We have recently found that AtGRP8 acts in concert with AtGRP7 to inﬂuence the transition to ﬂowering in A. thaliana (15). Apart from this, little is known about AtGRP8 function within the cell. Here we show that AtGRP8 forms a feedback loop that is interlocked with the AtGRP7 feedback loop. Recombinant AtGRP8 interacts with its own mRNA in vitro and promotes negative autoregulation in vivo by causing alternative splicing. The parts of the intron that are retained in the unproductively spliced transcripts show an exon-like evolutionary conservation, pointing to a functional role. As the alternatively spliced AtGRP8 and AtGRP7 transcripts are bona ﬁde NMD targets, it appears that the interlocked AtGRP7/AtGRP8 feedback loops harness autogenous alternative splicing-activated decay via the NMD pathway to ﬁne-tune the expression of their components. MATERIALS AND METHODS AtGRP8 overexpression in transgenic plants The protein-coding region of AtGRP8 was ampliﬁed by PCR from the cDNA with the upstream primer 50 GGCCATGGCTGAAGTTGAGT 30 and the down- stream primer 50 CCGGATCCTTTACCAGCCGCCAC CAC 30 covering the translation start and stop (bold) and comprising engineered NcoI and BamHI sites (under- lined), respectively. The PCR product was inserted between the CaMV 35S RNA promoter with the dupli- cated enhancer fused to the Tobacco Mosaic Virus omega translational enhancer and the CaMV polyadenylation signal (9). To express AtGRP8-RQ, the Arg47Gln muta- tion was introduced by PCR-mutagenesis (see below), sequenced and reinserted into the original plasmid by BglII/SacI digestion. The entire casettes were inserted into the binary vector pHPT1 that was obtained by replacing the promoter-less b-glucuronidase gene of pGPTV-HPT by the pUC19 polylinker (16). Arabidopsis thaliana L. Columbia plants were transformed by vacuum inﬁltration (17). Plant growth Seeds were germinated on one-half strength MS plates (18) containing 0.5% sucrose and the appropriate antibio- tic and grown in 16 h light/8 h dark cycles at a constant temperature of 208C. After 2 weeks, resistant plants were transferred to one-half strength MS plates without antibiotics. Recombinant GST-AtGRP8 and GST-AtGRP8-RQ Recombinant GST-AtGRP8 protein was constructed by inserting the AtGRP8 coding region into NotI-EcoRI- cut pGEX-6P1 vector (GE Healthcare, Freiburg, Germany). To generate the mutant variant GST-AtGRP8-RQ, Arg47 in GST-AtGRP8 was exchanged for glutamine by PCR with Phusion Polymerase (Finnzymes, Espoo, Finland) using the overlapping primers RQfor CGA GAGTGGAAGATCCCAAGGAT-TCGGATTCGTCA and RQrev TGACGAATCCGAATCCTTGGGATCTT CCACTCTCG. Silent mutations were introduced into neighbouring amino acids creating a diagnostic StyI site (underlined). The mutation was veriﬁed by sequencing. The recombinant proteins were expressed in E. coli BL21 DE3. Aﬃnity puriﬁcation by chromatography on Glutathione Sepharose (GE Healthcare) and concentra- tion of the eluate by centrifugation through Centricon 30 ﬁlter devices (Millipore, Billerica, MA, USA) were done as described (12,13). RNA-binding assay Synthetic oligoribonucleotides (ORN) were purchased from Biomers (Ulm, Germany). RNA bandshifts with recombinant AtGRP8 and the ORNs labelled at the 50 end with g-[32P] ATP were performed as previously described (12). To determine binding aﬃnities, 50 fmol of labeled ORN-binding substrates were incubated with increasing amounts of recombinant protein, respectively, in the presence of 1 mg tRNA. Bound and free RNA were quantiﬁed and Kd values calculated based on three inde- pendent experiments as described (12). RNA analysis Isolation of total RNA from Arabidopsis plants and hybridization of RNA gel blots with the gene-speciﬁc AtGRP7 and AtGRP8 probes were performed as described (8,19). Semiquantitative RT–PCR on retrotranscribed total RNA was done as described (15). Primers are listed in Table S2. Immunoblot analysis Protein extraction from Arabidopsis plants and incuba- tion of protein gel blots with antipeptide antibodies raised against AtGRP8 and AtGRP7, followed by chemi- luminescence detection were done as described (12). Secondary structure prediction Evolutionary conserved RNA secondary structure ele- ments were determined with the RNA-Decoder program (20,21). RNA-Decoder takes as input a ﬁxed alignment