Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process.     

The C-terminal zinc finger domain of Arabidopsis cold shock domain proteins is important for RNA chaperone activity during cold adaptation

Among the four cold shock domain proteins (CSDPs) identified in Arabidopsis thaliana, it has recently been shown that CSDP1 harboring seven CCHC-type zinc fingers, but not CSDP2 harboring two CCHC-type zinc fingers, function as a RNA chaperone during cold adaptation. However, the structural features relevant to this differing RNA chaperone activity between CSDP1 and CSDP2 remain largely unknown. To determine which structural features are necessary for the RNA chaperone activity of the CSDPs, the importance of the N-terminal cold shock domain (CSD) and the C-terminal zinc finger glycine-rich domains of CSDP1 and CSDP2 were assessed. The results of sequence motif-swapping and deletion experiments showed that, although the CSD itself harbored RNA chaperone activity, the number and length of the zinc finger glycine-rich domains of CSDPs were crucial to the full activity of the RNA chaperones. The C-terminal domain itself of CSDP1, harboring seven CCHC-type zinc fingers, also has RNA chaperone activity. The RNA chaperone activity and nuclei acid-binding property of the native and chimeric proteins were closely correlated with each other. Collectively, these results indicate that a specific modular arrangement of the CSD and the zinc finger domain determines both the RNA chaperone activity and nucleic acid-binding property of CSDPs; this, in turn, contributes to enhanced cold tolerance in plants as well as in bacteria.     

The RNA Chaperone and Protein Chaperone Activity of Arabidopsis Glycine-Rich RNA-Binding Protein 4 and 7 is Determined by the Propensity for the Formation of High Molecular Weight Complexes

RNA chaperones and protein chaperones are cellular proteins that can aid the correct folding of target RNAs and proteins, respectively. Although many proteins possessing RNA chaperone or protein chaperone activity have been demonstrated in diverse organisms, report evaluating the RNA chaperone and protein chaperone activity of a given protein is severely limited. Here, two glycine-rich RNA-binding proteins in Arabidopsis thaliana (AtGRPs), AtGRP7 exhibiting RNA chaperone activity and AtGRP4 exhibiting no RNA chaperone activity, were investigated for their protein chaperone activity. The heat-induced thermal aggregation of a substrate protein was significantly decreased with the addition of AtGRP4 depending on protein concentration, whereas the thermal aggregation of a substrate protein was further increased with the addition of AtGRP7, demonstrating that AtGRP4 but not AtGRP7 possesses protein chaperone activity. Size exclusion chromatography and electron microscopy analyses revealed that the formation of high molecular weight (HMW) complexes is closely related to the protein chaperone activity of AtGRP4. Importantly, the additional 25 amino acids at the N-terminus of AtGRP4 are crucial for HMW complex formation and protein chaperone activity. Taken together, these results show that the formation of HMW complexes is important for determining the RNA chaperone and protein chaperone activity of AtGRP4 and AtGRP7.     

Diverse roles of glycine-rich RNA-binding protein 7 in the response of camelina (<Emphasis Type="Italic">Camelina sativa</Emphasis>) to abiotic stress

Camelina sativa L. is an oilseed crop used as a potential low-cost biofuel resource. Despite the economic and agricultural benefits of this crop, studies demonstrating the physiological and genetic response of camelina to changing environmental conditions are limited. In this study, three stress-responsive glycine-rich RNA-binding proteins (GRPs) in camelina—named CsGRP7a, CsGRP7b, and CsGRP7c—were isolated, and their functional roles in stress responses were characterized. The three CsGRP7 genes had similar nucleotide and deduced amino acid sequences, and contained an N-terminal RNA-recognition motif and a C-terminal glycine-rich region. The CsGRP7 genes were ubiquitously expressed in all plant tissues, and CsGRP7 proteins were localized to both the cytoplasm and the nucleus. The expression of CsGRP7 genes was markedly upregulated by cold stress, whereas their expression was only slightly affected by salt or dehydration stress. Analysis of CsGRP7a-expressing transgenic Arabidopsis thaliana and camelina plants revealed that CsGRP7a plays a positive role in cold stress tolerance, but a negative role in salt or drought stress tolerance. All three CsGRP7s harbored RNA chaperone activity. Collectively, these data indicate that the stress-responsive CsGRP7s harbor RNA chaperone activity and play different roles in the plant response to abiotic stresses.     

Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins

The rbp gene family of the cyanobacterium Anabaena variabilis strain M3 consists of eight members that encode small RNA-binding proteins containing a single RNA recognition motif (RRM). Similar genes are found in the genomes of Synechocystis sp. PCC6803, Helicobacter pylori and Treponema pallidum, but are absent from the other completely sequenced prokaryotic genomes. The expression of the rbp genes of Anabaena is induced by low temperature, with the exception of the rbpD gene. We found four stretches of conserved sequences in the 5'-untranslated region of the cyanobacterial rbp genes that are known to be induced by low temperature. The cold-regulated Rbp proteins contain a short C-terminal glycine-rich domain. In this respect, these proteins are similar to plant and mammalian glycine-rich RNA-binding proteins (GRPs), which also contain a single RRM domain with a C-terminal glycine-rich domain and are highly expressed at low temperature. Detailed phylogenetic analysis showed, however, that the cyanobacterial Rbp proteins and the eukaryotic GRPs do not belong to a single lineage, but that the glycine-rich domains are likely to have been added independently. The cold-regulation of both types of proteins is also likely to have evolved independently. Furthermore, the chloroplast RNA-binding proteins are not likely to have originated from the Rbp proteins of endosymbiont cyanobacterium, but are supposed to have diverged from the GRPs. These results suggest that the cyanobacterial Rbp proteins and the eukaryotic GRPs are similar in both structure and regulation, but that this apparent similarity has resulted from convergent evolution.     

Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function

Glycine-rich RNA-binding proteins (GR-RBPs) are involved in cold shock response of plants as RNA chaperones facilitating mRNA transport, splicing and translation. GR-RBPs are bipartite proteins containing a RNA recognition motif (RRM) followed by a glycine-rich region. Here, we studied the structural basis of nucleic acid binding of full-length Nicotiana tabacum GR-RBP1. NMR studies of NtGR-RBP1 show that the glycine-rich domain, while intrinsically disordered, is responsible for mediating self-association by transient interactions with its RRM domain (NtRRM). Both NtGR-RBP1 and NtRRM bind specifically and with low micromolar affinity to RNA and single-stranded DNA. The solution structure of NtRRM shows that it is a canonical RRM domain. A HADDOCK model of the NtRRM–RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues. Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM. Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function.     

AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development

The glycine-rich protein AtGRP2 is one of the four members of the cold-shock domain (CSD) protein family in Arabidopsis. It is characterized by the presence of a nucleic acid-binding CSD domain, two glycine-rich domains and two CCHC zinc-fingers present in nucleic acid-binding proteins. In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene. Here, we demonstrate that AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development with a possible function in cold-response. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Down-regulation of AtGRP2 gene, using gene-silencing techniques resulted in early flowering, altered stamen number and affected seed development. A possible role of AtGRP2 as an RNA chaperone is discussed.     

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4°C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.     

Functional diversity of the plant glycine-rich proteins superfamily

The first plant glycine-rich proteins (GRPs) have been isolated more than 20 years ago based on their specific expression pattern and/or modulation by several biotic and abiotic factors. This superfamily is characterized by the presence of a glycine-rich domain arranged in (Gly)_n-X repeats. The presence of additional motifs, as well as the nature of the glycine repeats, groups them in different classes. The diversity in structure as well as in expression pattern, modulation and sub cellular localization have always indicated that these proteins, although classified as members of the same superfamily, would perform different functions in planta. Only now, two decades later, with the first functional characterizations of plant GRPs their involvement in diverse biological and biochemical processes are being uncovered. Here, we review the so far ascribed functions of plant GRPs.Key words: glycine-rich protein, cold-shock protein, RNA-binding protein, plant defense, flowering, cell elongation, RNA chaperone, signal transduction, oleosin, pollen competition     

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.