Control of Alternative Splicing by SICKLE/WARP2 is Required for Adaptation of the Plant Circadian Clock to Cool Temperatures

The circadian clock is a key mechanism for plants to anticipate and respond to daily and seasonal cycles of light and temperature. Cues of temperature and light cycles set the timing of circadian rhythms through the process of entrainment, yet the period length of rhythms remains constant because of temperature compensation. The molecular pathways that sense and respond to temperature are not well understood in plants. Recent studies in Arabidopsis thaliana suggest that alternative splicing of circadian clock transcripts is a fundamental mechanism that links ambient temperature perception with circadian clock rhythms. Splice variants of circadian clock transcripts are observed in response to temperature changes in the environment, but the source of splice variants and their effect on the circadian clock are not known. In addition, the core circadian clock genes LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) play an important role in modifying the circadian clock’s response to temperature.Here, we describe characterization of a new mutant allele of the Arabidopsis SICKLE (SIC) gene, sic-3, and the existing sic-1 allele with respect to their effects on the Arabidopsis circadian clock. sic was first identified as, warm acute response of PRR7 (warp2). sic has a unique temperature-specific defect in circadian clock function and in alternative splicing of circadian clock transcripts. sic exhibits low-amplitude or arrhythmic circadian rhythms under cool ambient temperature cycles, but not under light-dark entrainment. sic mutants also lengthen free running period in a manner consistent with impaired temperature compensation. sic mutant alleles accumulate splice variants of core circadian clock gene transcripts, including LHY and CCA1, which is exacerbated by cool temperatures. sic mutant alleles also modify transcription of circadian clock genes, particularly in cool temperatures. The cca1 lhy double mutant is epistatic to sic, indicating that control of LHY and CCA1 splicing by SIC is needed for circadian clock function. Furthermore, the double and triple mutant combinations of sic- 3 with cca1-1 and lhy-20 indicate that CCA1 and LHY function redundantly to modify temperature compensation, and that this regulation is dependent on SIC in cool temperatures. Finally, expression of LHY protein is altered in sic mutants in a temperature-dependent manner.SIC is a nuclear and cytoplasmic protein of unknown function, previously implicated in the control of alternative splicing, microRNA (miRNA) biogenesis, and stress responses (Zhan et al. 2012). SIC has homologous proline/serine rich proteins present only in angiosperms. The SIC protein interacts with spliceosome-associated proteins, including protein arginine methyltransferases PRMT4a and PRMT4b, as well as members of the NineTeen Complex (NTC), including the lariat debranching enzymeDBR1. The NTC is a multiprotein complex involved in transcription, transcription coupled DNA repair, and alternative splicing. This suggests that SIC maintains spliceosome efficiency at low temperatures through interaction with splicing factors, and this aspect of pre-RNA metabolism is critical for low temperature adaptation and for the circadian clock’s response to temperature cues.We propose the following model: alternative splicing converts circadian clock pre- mRNA transcripts to mature mRNA in a temperature-dependent manner, and the resulting splice variant pool determines the proper accumulation and timing of circadian clock proteins, especially LHY and CCA1. This form of post-transcriptional circadian clock regulation is crucial for temperature compensation and thermocycle entrainment in Arabidopsis. Where SIC fits into this model is unknown, but our data suggests two possibilities: 1) SIC modifies PRMT4a and PRMT4b activity, which then affects spliceosome activity, and/or 2) SIC modifies the NTC to regulate transcription, transcription coupled DNA repair, and alternative splicing. All of these processes are temperature-dependent, and it is clear that the activity of SIC is most important in cool temperatures. SIC’s affect on LHY and CCA1 alternative splicing appears to play a large role in regulating temperature compensation at cool temperatures. Discovery of the biochemical and molecular activities of SIC is certain to reveal important details about the functional link between alternative splicing and temperature responses within the Arabidopsis circadian clock, and holds the potential to provide new insights into the molecular processes involved in pre-mRNA processing