Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.     

Arginine methylation of the cellular nucleic acid binding protein does not affect its subcellular localization but impedes RNA binding

Cellular nucleic acid binding protein (CNBP) contains seven zinc finger (ZF) repeats and an arginine and glycine (RG) rich sequence between the first and the second ZF. CNBP interacts with protein arginine methyltransferase PRMT1. Full-length but not RG-deleted or mutated CNBP can be methylated. Treatment with a methylation inhibitor AdOx reduced CNBP methylation, but did not affect the concentrated nuclear localization of CNBP. Nevertheless, arginine methylation of CNBP appeared to interfere with its RNA binding activity. Our findings show that arginine methylation of CNBP in the RG motif did not change the subcellular localization, but regulated its RNA binding activity.Structured summary of protein interactionsPRMT1 binds to CNBP by pull down (View interaction)PRMT1 methylates CNBP by enzymatic study (View interaction)CNBP physically interacts with PRMT1 by anti tag coimmunoprecipitation (View interaction)     

Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients

Background

Antibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). Anti-Sm autosera have been reported to specifically recognize Sm D1 and D3 with symmetric di-methylarginines (sDMA). We investigated if anti-Sm sera from local SLE patients can differentially recognize Sm proteins or any other proteins due to their methylation states.

Results

We prepared HeLa cell proteins at normal or hypomethylation states (treated with an indirect methyltransferase inhibitor adenosine dialdehyde, AdOx). A few signals detected by the anti-Sm positive sera from typical SLE patients decreased consistently in the immunoblots of hypomethylated cell extracts. The differentially detected signals by one serum (Sm1) were pinpointed by two-dimensional electrophoresis and identified by mass spectrometry. Three identified proteins: splicing factor, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein D-like (hnRNP DL) and cellular nucleic acid binding protein (CNBP) are known to contain methylarginines in their glycine and arginine rich (GAR) sequences. We showed that recombinant hnRNP DL and CNBP expressed in Escherichia coli can be detected by all anti-Sm positive sera we tested. As CNBP appeared to be differentially detected by the SLE sera in the pilot study, differential recognition of arginine methylated CNBP protein by the anti-Sm positive sera were further examined. Hypomethylated FLAG-CNBP protein immunopurified from AdOx-treated HeLa cells was less recognized by Sm1 compared to the CNBP protein expressed in untreated cells. Two of 20 other anti-Sm positive sera specifically differentiated the FLAG-CNBP protein expressed in HeLa cells due to the methylation. We also observed deferential recognition of methylated recombinant CNBP proteins expressed from E. coli by some of the autosera.

Conclusion

Our study showed that hnRNP DL and CNBP are novel antigens for SLE patients and the recognition of CNBP might be differentiated dependent on the level of arginine methylation.     

Model Organisms: New Insights: Into Ion Channel and Transporter Function. Caenorhabditis elegans ClC-type chloride channels: novel variants and functional expression

Six ClC-type chloridechannel genes have been identified in Caenorhabditiselegans, termed clh-1 through clh-6. cDNAsequences from these genes suggest that clh-2,clh-3, and clh-4 may code for multiple channelvariants, bringing the total to at least nine channel types in thisnematode. Promoter-driven green fluorescent protein (GFP) expression intransgenic animals indicates that the protein CLH-5 isexpressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells,and the remaining isoforms vary from those restricted to a single cellto those expressed in over a dozen cells of the nematode. In an Sf9cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did notform functional plasma membrane channels. In contrast, both CLH-1 andCLH-3b produced strong, inward-rectifying chloride currents similar tothose arising from mammalian ClC2, but which operate over differentvoltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene familyprovides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.

     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Construction of a Contiguous 874-kb Sequence of the Escherichia coli-K12 Genome Corresponding to 50.0-68.8 min on the Linkage Map and Analysis of Its Sequence Features

The contiguous 874.423 base pair sequence corresponding to the50.0–68.8 min region on the genetic map of the Escherichiacoli K-12 (W3110) was constructed by the determination of DNAsequences in the 50.0–57.9 min region (360 kb) and twolarge (100 kb in all) and five short gaps in the 57.9–68.8min region whose sequences had been registered in the DNA databases.We analyzed its sequence features and found that this regioncontained at least 894 potential open reading frames (ORFs),of which 346 (38.7%) were previously reported, 158 (17.7%) werehomologous to other known genes, 232 (26.0%) were identicalor similar to hypothetical genes registered in databases, andthe remaining 158 (17.7%) showed no significant similarity toany other genes. A homology search of the ORFs also identifiedseveral new gene clusters. Those include two clusters of fimbrialgenes, a gene cluster of three genes encoding homologues ofthe human long chain fatty acid degradation enzyme complex inthe mitochondrial membrane, a cluster of at least nine genesinvolved in the utilization of ethanolamine, a cluster of thesecondary set of 11 hyc genes participating in the formate hydrogenlyasereaction and a cluster of five genes coding for the homologuesof degradation enzymes for aromatic hydrocarbons in Pseudomonasputida. We also noted a variety of novel genes, including twoORFs, which were homologous to the putative genes encoding xanthinedehydrogenase in the fly and a protein responsible for axonalguidance and outgrowth of the rat, mouse and nematode. An isoleucinetRNA gene, designated ileY , was also newly identified at 60.0min.     

Abscisic acid regulates TSRF1-mediated resistance to Ralstonia solanacearum by modifying the expression of GCC box-containing genes in tobacco

Although recent studies have established a significant regulatoryrole for abscisic acid (ABA) and ethylene response factor (ERF)proteins in plant pathogen resistance, it is not clear whetherand how ABA performs this role. Previously, it was reportedthat an ERF protein, TSRF1, activates the expression of GCCbox-containing genes and significantly enhances the resistanceto Ralstonia solanacearum in both tobacco and tomato plants.Here, it is reported that TSRF1-regulated pathogen resistanceis modified by ABA application. TSRF1 activates the expressionof ABA biosynthesis-related genes, resulting in the increaseof ABA biosynthesis, which further stimulates ethylene production.More interestingly, ABA application decreases, while the inhibitorof ABA biosynthesis fluridone increases, the TSRF1-enhancedresistance to R. solanacearum. This observation is further supportedby the finding that ABA and fluridone reversibly modify theability of TSRF1 to bind the ethylene-responsive GCC box, consequentlyaltering the expression of element-controlled genes. These resultstherefore establish that TSRF1-regulated resistance to R. solanacearumcan be modified in tobacco by ABA. Key words: Abscisic acid, ERF protein TSRF1, GCC box-containing genes, Ralstonia solanacearum, tobacco     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

Chemical Patterns, Compartments and a Binary Epigenetic Code in Drosophila

I propose a model which accounts for the geometries and sequencein which compartmental boundary lines arise on the differentimaginal discs, and on the blastoderm of Drosophila melanogaster;and propose that successive lines are recorded by differentbinary switches, to create a binary epigenetic code word specifyingeach disc, and disc compartment. I suppose a biochemical systemundergoing reaction and diffusion acts throughout development.As an imaginal disc grows, a succession of differently shapedchemical concentration patterns form at a discrete set of discsizes. I suppose a specific concentration of one chemical isa threshold. Concentrations above or below threshold switchcells to one or another of two commitments. Then the line acrossthe imaginal disc with the threshold concentration is a predictedcompartmental boundary. The sequence and geometries of suchlines predict the compartmental boundaries seen on the wingdisc, the other discs, and on the blastoderm stage egg. Thecompartmental lines on the wing disc suggest that a terminalcompartment is specified by a combination of binary names recordinga sequence of binary commitments: anterior, not posterior; dorsal,not ventral; wing, not thorax; proximal, not distal. Each combinationcomprises a binary epigenetic code word. Recently I constructedan independent model for transdetermination in Drosophila whichproposed a similar binary epigenetic code for the differentdiscs. The clone restriction lines predicted on the blastodermby my transdetermination model, the chemical pattern model,and analogy with the wing disc, are nearly identical. Severalare already confirmed. The resultant binary code scheme correctlypredicts many relative transdetermination frequencies and accountssimply for the action of most homeotic mutants as genes whichalter a single switch state in one or more discs.     

Isolation of Genes for Low-Temperature-Induced Proteins in Rice by a Simple Subtractive Method

We have isolated cDNAs that correspond to three distinct low-temperature-inducedmRNAs from rice cells using an effective, simple subtractionmethod. The corresponding genes were designated lip (gene encodinglow-temperature-induced protein) –5,–9 and –19.The expression of these genes was slightly stimulated lip5 andlip19) or unaffected (lip9) by abscisic acid, unlike the expressionof the rabl6A-D genes which are readily induced by either abscisicacid or high osmotic stress. Time-course analysis revealed thatwhile the lip5 gene was induced 3 h after temperature down-shift,and the lip9 and lipl9 genes were induced 6 h after such a shift. ¹Present address: Division of Molecular Medicine, Clinical ResearchCentre, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ,U.K. (Received June 29, 1991; Accepted October 7, 1991)     

A gene encoding a protein with seven zinc finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe.

Byr3 was selected as a multicopy suppressor of the sporulation defects of diploid Schizosaccharomyces pombe cells that lack ras1. Like cells mutant at byr1 and byr2, two genes that encode putative protein kinases and that in multiple copies are also suppressors of the sporulation defects of ras1 null diploid cells, cells mutant at byr3 are viable but defective in conjugation. Nucleic acid sequence indicates byr3 has the capacity to encode a protein with seven zinc finger binding domains, similar in structure to the cellular nucleic acid binding protein (CNBP), a human protein that was identified on the basis of its ability to bind DNA. Expression of CNBP in yeast can partially suppress conjugation defects of cells lacking byr3.