New Hampshire 03755 (P.A.S., C.R.M.)Transcriptional and posttranscriptional regulation are well-established mechanisms for circadian gene expression. Among the latter, differential messenger RNA (mRNA) stability has been hypothesized to control gene expression in response to the clock. However, direct proof that the rate of mRNA turnover can be regulated by the clock is lacking. Previous microarray expression data for unstable mRNAs in Arabidopsis (Arabidopsis thaliana) revealed that mRNA instability is associated with a group of genes controlled by the circadian clock. Here, we show that CCR-LIKE (CCL) and SENESCENCE ASSOCIATED GENE 1 transcripts are differentially regulated at the level of mRNA stability at different times of day. In addition, the changes in CCL mRNA stability continue under free-running conditions, indicating that it is controlled by the Arabidopsis circadian clock. Furthermore, we show that these mRNAs are targets of the mRNA degradation pathway mediated by the downstream (DST) instability determinant. Disruption of the DST-mediated decay pathway in the dst1 mutant leads to aberrant circadian mRNA oscillations that correlate with alterations of the half-life of CCL mRNA relative to parental plants in the morning and afternoon. That this is due to an effect on the circadian control is evidenced by mRNA decay experiments carried out in continuous light. Finally, we show that the defects exhibited by dst mutants are reflected by an impact on circadian regulation at the whole plant level. Together, these results demonstrate that regulation of mRNA stability is important for clock-controlled expression of specific genes in Arabidopsis. Moreover, these data uncover a connection between circadian rhythms and a sequence-specific mRNA decay pathway.Plants, like many other organisms, have internal clocks that command biological rhythms with a period close to 24 h. These rhythms provide selective advantages because they allow anticipation of the daily changes in environmental conditions (Ouyang et al., 1998;Green et al., 2002). Examples of processes that can exhibit circadian rhythms in plants are leaf movement, hypocotyl elongation, stomatal opening, and floral induction (for review, see McClung, 2001). At the molecular level, DNA microarray experiments have shown that 2% to 6% of Arabidopsis (Arabidopsis thaliana) mRNAs can oscillate (Harmer et al., 2000;Schaffer et al., 2001). For Arabidopsis and other circadian clocks studied to date, the core circadian oscillator is comprised of transcriptional feedback loops. The underlying master oscillator in Arabidopsis is believed to include the LATE ELONGATED HYPO-COTYL (LHY; Schaffer et al., 1998), the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1; Wang and Tobin, 1998), and the TIMING OF CAB EXPRESSION 1 (TOC1; Millar et al., 1995;Strayer et al., 2000) genes. Similar to what is observed in other systems (Glossop et al., 1999), the clock components form a regulatory loop in which TOC1 positively regulates LHY and CCA1 gene expression, and LHY and CCA1 proteins in turn...
One of the ways a cell can rapidly and tightly regulate gene expression is to target specific mRNAs for rapid decay. A number of mRNA instability sequences that mediate rapid mRNA decay have been identified, particularly from multicellular eukaryotes, but pinpointing the cellular components that play critical roles in sequence-specific decay in vivo has been more difficult. In contrast, general pathways of mRNA degradation in yeast have been well established through the analysis of mutants affecting the general mRNA decay machinery. Strategies to isolate mutants in sequencespecific mRNA decay pathways, although extremely limited so far, have the potential to be just as powerful. In the study reported here, a selection in transgenic plants allowed the isolation of rare mutants of Arabidopsis thaliana that elevate the abundance of mRNAs that contain the plant mRNA instability sequence called DST (downstream element). This instability sequence is highly conserved in unstable small auxin up RNA (SAUR) transcripts. Genetic analysis of two dst mutants isolated via this selection showed that they are incompletely dominant and represent two independent loci. In addition to affecting DST-containing transgene mRNAs, mutations at both loci increased the abundance of the endogenous DST-containing SAUR-AC1 mRNA, but not controls lacking DST sequences. That these phenotypes are caused by deficiencies in DST-mediated mRNA decay was supported by mRNA stability measurements in transgenic plants. Isolation of the dst mutants provides a means to study sequence-specific mRNA degradation in vivo and establishes a method to isolate similar mutants from other organisms.
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