Daniel Alejandro Careno

The Arabidopsis transcriptome must be continuously reshaped to integrate endogenous and environmental cues, including the circadian clock and ambient light. This reprogramming relies on the coordinated synthesis and degradation of mRNAs, yet the contribution of RNA decay factors to circadian regulation remains poorly characterized. Here, we show that nearly all core clock genes are high-amplitude clock-controlled genes (HA-CCGs). A meta-analysis of transcriptome-wide mRNA decay datasets revealed that HA-CCGs have consistently shorter half-lives across multiple studies. We further identify the mRNA decapping activator DECAPPING PROTEIN 5 (DCP5) as a key regulator of circadian RNA stability. Using iCLIP, we show that DCP5 binds preferentially to HA-CCG transcripts at AU-rich motifs in their 3′ UTRs, thereby modulating their stability. Accordingly, the dcp5 mutant displays an extended circadian period at both physiological and molecular levels. Our findings establish mRNA decay as a post-transcriptional regulatory layer that fine-tunes the decay of multiple key transcripts within the circadian system.

Summary: Genetic variation at splice site signals significantly influences alternative splicing, leading to transcriptomic and proteomic diversity that enhances phenotypic plasticity and adaptation. However, novel splice variants can negatively impact gene expression and developmental stability. Canalization–the ability of an organism to maintain a consistent phenotype despite genetic or environmental variations–helps balance the effects of genetic variation on development and evolution. Protein arginine methyltransferase 5 (PRMT5) is a key splicing regulator in plants and animals. Most splicing changes in prmt5 mutants are linked to weak donor splice sites, suggesting that PRMT5 may buffer splicing against genetic variation. We examined PRMT5′s effects on splicing and development in two genetically divergent Arabidopsis thaliana accessions with different single nucleotide polymorphisms affecting donor splice sites. We found that PRMT5 inactivation significantly increased splicing and phenotypic differences between the accessions. Our findings suggest that PRMT5 contributes to canalization, mitigating the impact of splice site polymorphisms and facilitating the evolution of adaptive splicing patterns.

Light is both the main source of energy and a key environmental signal for plants. It regulates not only gene expression but also the tightly related processes of splicing and alternative splicing (AS). Two main pathways have been proposed to link light sensing with the splicing machinery. One occurs through a photosynthesis-related signal, and the other is mediated by photosensory proteins, such as red light-sensing phytochromes. Here, we evaluated the relative contribution of each of these pathways by performing a transcriptome-wide analysis of light regulation of AS in plants that do not express any functional phytochrome (phyQ). We found that an acute 2-h red-light pulse in the middle of the night induces changes in the splicing patterns of 483 genes in wild-type plants. Approximately 30% of these genes also showed strong light regulation of splicing patterns in phyQ mutant plants, revealing that phytochromes are important but not essential for the regulation of AS by R light. We then performed a meta-analysis of related transcriptomic datasets and found that different light regulatory pathways can have overlapping targets in terms of AS regulation. All the evidence suggests that AS is regulated simultaneously by various light signaling pathways, and the relative contribution of each pathway is highly dependent on the plant developmental stage.

Circadian rhythms enable organisms to anticipate and adjust their physiology to periodic environmental changes. These rhythms are controlled by biological clocks that consist of a set of clock genes that regulate each other expression. Circadian oscillations in mRNA levels require regulation of mRNA production and degradation. While transcription factors controlling clock function have been well characterized from cyanobacteria to humans, the role of factors controlling mRNA decay is largely unknown. Here, we show that mutations in XRN4, the central component of the 5’-3’ mRNA decay pathway, alter clock function in Arabidopsis. We found that xrn4 mutants display long period phenotypes for clock gene expression and for the rhythm of leaf movement. These circadian defects were associated with changes in the circadian phases, but not overall mRNA levels, of several core clock genes. We then used non-invasive transcriptome-wide mRNA stability analysis to identify genes and pathways regulated by XRN4. Among genes affected in the xrn4 mutant at the transcriptional and post-transcriptional level, we found an enrichment in genes involved in auxin, ethylene, ABA signaling, and also circadian rhythmicity, although no significant effects were observed for canonical core-clock genes. Strikingly, the mRNAs of several clock regulated BBX genes were stabilized in xrn4 mutants. Some of these BBX genes are auxiliary factors controlling the pace of the clock and are candidates to mediate XRN4 effects on circadian period. Our results establish that, in Arabidopsis, the control of 5’-3’ mRNA decay by XRN4 constitutes a novel post-transcriptional regulatory layer of the circadian gene network.

Light signaling pathways interact with the circadian clock to help organisms synchronize physiological and developmental processes to periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Members of the family of NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED (LNK) genes play key roles linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana. Particularly, LNK1 and LNK2 were shown to control circadian rhythms, photomorphogenic responses, and photoperiod-dependent flowering time. Here we analyze the role of the four members of the LNK family in Arabidopsis in these processes. We found that depletion of the closely related LNK3 and LNK4 in a lnk1;lnk2 mutant background affects circadian rhythms, but not other clock-regulated processes such as flowering time and seedling photomorphogenesis. Nevertheless, plants defective in all LNK genes (lnkQ quadruple mutants) display developmental alterations that lead to increased rosette size, biomass, and enhanced phototropic responses. Our work indicates that members of the LNK family have both distinctive and partially overlapping functions, and are an essential link to orchestrate light-regulated developmental processes.