6984 Nucleic Acids Research, 2008, Vol. 36, No. 22 Figure 5. Diﬀerential regulation by cold of AtGRP8 and AtGRP7. (A) Col plants grown for 2 weeks in 16 h light/8 h dark cycles at 208C were transferred to 16-h light/8-h dark cycles at 48C and harvested on day 0, 1, 2, 4 and 7 at zt2 and zt10, respectively. (B) Immunoblots with total protein extracts from the same plants were probed with the AtGRP8 (A, top) and AtGRP7 (B, top) antibodies and an LHCP anti- body (A and B, bottom) as loading control. (C) Semiquantitative RT- PCR of AtGRP8. (D) Semiquantitative RT-PCR of AtGRP7. (E) Semiquantitative RT-PCR of eIF-4A as constitutive control. The expo- nential range was determined by comparing the signal with increasing number of cycles. The absence of genomic DNA was conﬁrmed with nonretrotranscribed RNA. Notably, the retained upstream part of the intron exhibits a higher degree of conservation than the downstream part spliced out in the as_AtGRP7 and as_AtGRP8 variants (Figure 7). A comparison of AtGRP8 with AtGRP7 and orthologs from mustard, oilseed rape, tobacco and Pelargonium shows that the degree of conservation reaches that of the surrounding exons (Figure 7, Table S1). The open reading frames contain an in frame termination codon within the ﬁrst half of the intron and code for predicted 5 kDa polypeptides comprising only the RNP2 moiety of the RRM. As intact RNP1 is important for high-aﬃnity RNA binding of AtGRP7 (12) and AtGRP8 (Table 2), these truncated polypeptides presum- ably do not interact with the RNA target sites in a pro- ductive manner but theoretically, they could interfere with AtGRP7 and AtGRP8 function. So far, however, these polypeptides have not been detected in Arabidopsis WT plants, the upf mutants or in Sinapis alba (data not shown). Thus, the conservation likely is not due to protein coding and therefore implies a regulatory function, e.g. to convey degradation when retained. A similar scenario has been observed for the ribosomal protein L12 in Caenorhabditis elegans (41). Elevated L12 levels promote the formation of an alternatively spliced transcript with a PTC due to removal of a partial intron. Thus, this autoregulatory circuit also couples unproductive splicing with destruction via the NMD path- way and, like for AtGRP7 and AtGRP8, the retained part Figure 6. Eﬀect of upf1 and upf3 mutations on steady-state abundance of the AtGRP8 and AtGRP7 pre-mRNAs. RNA from the upf1-5, upf3-1 and upf3-2 mutants and WT harvested at zt10 was reverse-transcribed. PCR ampliﬁcation of the AtGRP8 (A) and AtGRP7 pre-mRNA (B) was performed using speciﬁc primers and 24 cycles. The gel with the PCR products was blotted and hybridized with the AtGRP8 cDNA (A) or AtGRP7 cDNA (B). Ampliﬁcation with ACTIN primers served as control (C). of the intron displays a striking sequence conservation in worms (41). Notably, the AtGRP7 transcript oscillates with one of the highest amplitudes observed for circadian genes in Arabidopsis (19). Following transcriptional activation by the circadian clock during the day and a steep rise in mRNA level, the AtGRP7 mRNA needs to decay during the declining phase below a level attained by time-of-day dependent transcriptional control alone in order to prevent damping (19,35). One way to accomplish high- amplitude mRNA oscillations would be by severely decreasing mRNA half life during the course of the day (42). However, such a scenario would also impact non- clock functions of AtGRP7 and AtGRP8, as in addition to their regulation by the circadian clock AtGRP8 and in particular AtGRP7 respond to numerous external factors (28). Transcriptional activation by external regulatory cues presumably would not bring about suﬃciently high levels of a short- lived mRNA during a considerable part of the day to allow increase in protein steady-state abun- dance. Thus, AS-NMD elicited by the proteins themselves may be an eﬃcient way to adjust mRNA and conse- quently protein levels. Utilization of NMD for negative autoregulation has been widely observed for proteins involved in splicing reg- ulation. The mammalian SR protein SC35 downregulates its expression through alternative splicing, resulting in an unstable transcript (43). However, in contrast to as_AtGRP7 and as_AtGRP8, the alternative SC35 trans- cripts retain full coding capacity. Crossregulation recently has been described for the polypyrimidine tract-binding protein (PTB), a global repressor of alternative splicing in nonneuronal cells, and its neurally expressed paralogue nPTB. PTB induces skipping of its own exon 11, produ- cing an NMD substrate, and also induces exon-skipping in nPTB, resulting in a switch from PTB to nPTB expression during the development of neurons that impacts neural- speciﬁc splicing patterns of downstream targets. nPTB is able to autoregulate at the level of exon skipping when PTB is absent (44). Spellman showed that nPTB is able to repress PTB exon 11, however whether this occurs in vivo has not yet been determined (45). For AtGRP7 and AtGRP8 we have found autoregula- tion and reciprocal crossregulation, showing that the