Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.     

Circadian clock-regulated physiological outputs: Dynamic responses in nature

The plant circadian clock is involved in the regulation of numerous processes. It serves as a timekeeper to ensure that the onset of key developmental events coincides with the appropriate conditions. Although internal oscillating clock mechanisms likely evolved in response to the earth's predictable day and night cycles, organisms must integrate a range of external and internal cues to adjust development and physiology. Here we introduce three different clock outputs to illustrate the complexity of clock control. Clock-regulated diurnal growth is altered by environmental stimuli. The complexity of the photoperiodic flowering pathway highlights numerous nodes through which plants may integrate information to modulate the timing of flowering. Comparative analyses among ecotypes that differ in flowering response reveal additional environmental cues and molecular processes that have developed to influence flowering. We also explore the process of cold acclimation, where circadian inputs, light quality, and stress responses converge to improve freezing tolerance in anticipation of colder temperatures.     

Environmental and genetic effects on circadian clock-regulated gene expression in Arabidopsis.

Expression patterns of the cold-circadian rhythm-RNA binding (CCR) and chlorophyll a/b binding (CAB) protein genes have circadian rhythms with phases that are different from each other and are affected differently by cold (4 degrees C) treatment. Cycling of CCR and CAB RNA levels was observed in Arabidopsis seedlings grown for 5 days at 4 degrees C under a light/ dark photoperiod, although the cycling had reduced amplitude compared with normal growth conditions (20 degrees C). CCR RNA levels were elevated in the cold, whereas CAB RNA levels were reduced in the cold relative to levels in control seedlings. Cold pulses (4 degrees C for 12 or 20 hr) under continuous light affected the rhythms of CCR and CAB RNA levels in similar ways. The 12-hr cold pulse caused a 4-hr phase delay in both rhythms, whereas the 20-hr cold pulse resulted in a 12-hr phase delay in both rhythms. The timing of CAB expression 1 (toc1) mutation shortened the period of the CCR rhythm, matching previous results for the regulation of the CAB-luciferase (CAB-luc) transgene in this mutant. The results suggest that CCR and CAB share clock machinery but are regulated by downstream components that are affected differently by the cold. Also, the circadian clock regulating these genes in Arabidopsis has a cold-sensitive phase under continuous light conditions.     

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.     

Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana

Journal of Plant Research - The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the...