Evaluation and classification of RING-finger domains encoded by the <Emphasis Type="Italic">Arabidopsis</Emphasis> genome

Background

In computational analysis, the RING-finger domain is one of the most frequently detected domains in the Arabidopsis proteome. In fact, it is more abundant in Arabidopsis than in other eukaryotic genomes. However, computational analysis might classify ambiguous domains of the closely related PHD and LIM motifs as RING domains by mistake. Thus, we set out to define an ordered set of Arabidopsis RING domains by evaluating predicted domains on the basis of recent structural data.

Results

Inspection of the proteome with a current InterPro release predicts 446 RING domains. We evaluated each detected domain and as a result eliminated 59 false positives. The remaining 387 domains were grouped by cluster analysis and according to their metal-ligand arrangement. We further defined novel patterns for additional computational analyses of the proteome. They were based on recent structural data that enable discrimination between the related RING, PHD and LIM domains. These patterns allow us to predict with different degrees of certainty whether a particular domain is indeed likely to form a RING finger.

Conclusions

In summary, 387 domains have a significant potential to form a RING-type cross-brace structure. Many of these RING domains overlap with predicted PHD domains; however, the RING domain signature mostly prevails. Thus, the abundance of PHD domains in Arabidopsis has been significantly overestimated. Cluster analysis of the RING domains defines groups of proteins, which frequently show significant similarity outside the RING domain. These groups document a common evolutionary origin of their members and potentially represent genes of overlapping functionality.

     

AtMBD6, a methyl CpG binding domain protein,maintains gene silencing in <Emphasis Type="Italic">Arabidopsis</Emphasis> by interacting with RNA binding proteins

DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

NMR study of non-structural proteins–part III: <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and side-chain resonance assignment of macro domain from <Emphasis Type="Italic">Chikungunya</Emphasis> virus (CHIKV)

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130–190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.     

Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) JAZ3 and SLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation

Jasmonate ZIM-domain (JAZ) proteins and DELLA proteins are key negative regulators of jasmonates (JAs) and gibberellin (GA) signaling, respectively. In this study, we found JA and GA synergistically promote fiber cell initiation. We characterized the cellular function of a JAZ protein (GhJAZ3), and a DELLA protein (GhSLR1) of cotton (Gossypium hirsutum). GhJAZ3 is specifically expressed in elongating fibers, while GhSLR1 is expressed in different tissues and at a relatively higher level in 3 DPA ovules. GhSLR1 and GhJAZ3 proteins are localized in the cell nucleus. Yeast two-hybrid analysis indicated that GhSLR1, GhJAZ3 and GhDEL65 could interact with each other, and GhSLR1 could also interact with GhBZR1. Overexpression of GhJAZ3 in Arabidopsis increased hypocotyl and root length, leaf trichome length, and plant height, but decreased the number of leaf trichome, while overexpression of GhSLR1 in Arabidopsis decreased hypocotyl length, leaf trichome length and density. Expression of several leaf trichome initiation determinators (GL3, GL2, TTG2 and MYB23) was down-regulated in GhJAZ3 or GhSLR1 transgenic Arabidopsis, while expression of the cell elongation related genes (EXP1, EXP8, EXPL2 and XTH4) was altered in the GhJAZ3 and GhSLR1 transgenic Arabidopsis. Taken together, these results demonstrate that GhJAZ3 and GhSLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation.     

Identification of Ribosomal Protein L1-Binding Sites in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis> mRNAs

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.     

Chromosomal distribution of H3K4me2, H3K9me2 and 5-methylcytosine: variations associated with polyploidy and hybridization in <Emphasis Type="Italic">Brachiaria</Emphasis> (Poaceae)

Key message

Assessment of chromosomal distribution of modified histones and 5-methylcytosine shown that there are diversification of chromosomal types among species of Brachiaria and its interspecific hybrids.

Abstract

Histone post-translational modifications and DNA methylation are epigenetic processes that are involved in structural and functional organization of the genome. This study compared the chromosomal distribution of modified histones and 5-methylcytosine (5-mCyt) in species and interspecific hybrids of Brachiaria with different ploidy levels and reproduction modes. The relation between H3K9me2 and 5-mCyt was observed in the nucleolus organizer region, centromeric central domain and pericentromeric region. H3K4me2 was detected in euchromatic domains, mainly in the terminal chromosomal regions. Comparison of chromosomal distribution among species and hybrids showed greater variation of chromosomal types for the H3K9me2 in B. decumbens (tetraploid and apomictic species) and the 963 hybrid, while, for the H3K4me2, the variation was higher in B. brizantha and B. decumbens (tetraploid and apomictic species) and 963 hybrid. The chromosome distribution of 5-mCyt was similar between B. brizantha and B. decumbens, which differ from the distribution observed in B. ruziziensis (diploid and sexual species). Significant alterations in DNA methylation were observed in the artificially tetraploidized B. ruziziensis and in the interspecific hybrids, possibly as result of hybridization and polyploidization processes. The monitoring of histone modifications and DNA methylation allowed categorizing nuclear and chromosomal distribution of these epigenetic marks, thus contributing to the knowledge of composition and structure of the genome/epigenome of Brachiaria species and hybrids. These data can be useful for speciation and genome evolution studies in genus Brachiaria, and represent important markers to explore relationships between genomes.

     

Characterization of the inner membrane protein BB0173 from <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background

The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3β1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made.

Results

Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions.

Conclusions

Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.