Tracking the elusive 5′ exonuclease activity of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> RNase J

Key message

Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.

Abstract

RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

     

Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter P _tac and primers ensuring randomization of the four central nucleotides in the ?35 region. DNA fragments containing the tac-like promoters and a selective marker (Cm ^R) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 4⁴ = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 10² Miller units with the weakest promoter to 10⁴ Miller units with consensus P _tac Sequencing of the modified promoters showed that randomization of three positions in the ?35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.     

Crystal structure of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> RraAS2, an unusual member of the RNase E inhibitor RraA protein family

Bacterial ribonuclease E (RNase E) plays a crucial role in the processing and decay of RNAs. A small protein named RraA negatively regulates the activity of RNase E via protein-protein interaction in various bacteria. Recently, RraAS1 and RraAS2, which are functional homologs of RraA from Escherichia coli, were identified in the Gram-positive species Streptomyces coelicolor. RraAS1 and RraAS2 inhibit RNase ES ribonuclease activity in S. coelicolor. RraAS1 and RraAS2 have a C-terminal extension region unlike typical bacterial RraA proteins. In this study, we present the crystal structure of RraAS2, exhibiting a hexamer arranged in a dimer of trimers, consistent with size exclusion chromatographic results. Importantly, the C-terminal extension region formed a long α-helix at the junction of the neighboring subunit, which is similar to the trimeric RraA orthologs from Saccharomyces cerevisiae. Truncation of the C-terminal extension region resulted in loss of RNase ES inhibition, demonstrating its crucial role. Our findings present the first bacterial RraA that has a hexameric assembly with a C-terminal extension α-helical region, which plays an essential role in the regulation of RNase ES activity in S. coelicolor.     

Biosynthesis of secreted ribonucleases of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> and <Emphasis Type="Italic">Bacillus circulans</Emphasis> under nitrogen starvation

The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

Phylogenetic Diversity of the Sulfur Cycle Bacteria in the Bottom Sediments of the Chersonesus Bay

The Black Sea is the largest meromictic basin, in the bottom sediments of which a powerful biogenic process of sulfide production occurs. The goal of the present work was to obtain data on phylogenetic diversity of the sulfur cycle microorganisms (sulfate-reducing and sulfur-oxidizing bacteria) in the Black Sea coastal gas-saturated bottom sediments. The samples were collected in the Chersonesus (Blue) Bay near Sevastopol from whitish bacterial mats of sulfurettes, and from the upper layer of the nearby seabed. Using DNA isolated from the native samples and obtained enrichment cultures, PCR analysis was performed with oligonucleotide primers specific to the fragments of the 16S rRNA genes of the main subgroups of sulfatereducing bacteria (SRB) and to the fragments of the dsrB gene (both reductive and oxidative types), encoding the β-subunit of dissimilatory (bi)sulfite reductase, the key enzyme in the sulfur cycle, inherent in both sulfate- reducing and sulfur-oxidizing microorganisms. The presence of 16S rRNA gene fragments specific to the genera Desulfobacterium, Desulfobacter, Desulfococcus–Desulfonema–Desulfosarcina, and Desulfovibrio–Desulfomicrobium was detected in the DNA samples isolated from coastal bottom bacterial mats. Usage of denaturing gradient gel electrophoresis (DGGE) with subsequent sequencing of reamplified dsrB gene fragments revealed that according to deduced amino acid sequences encoded by the dsrB gene (reductive type), SRB from the coastal gas-saturated bottom sediments of the Black Sea had the highest homology (92?99%) with the dsrB gene of cultured SRB belonging to the genera Desulfovibrio, Desulfatitalea, Desulfobacter, and Desulfobacterium, as well as with uncultured SRB strains from various marine habitats, such as bottom sediments of the Northern and Japanese seas. Deduced amino acid sequences encoded by the oxidative dsrB gene had the highest homology (90?99%) with the relevant sequences of the genera Thiocapsa, Thiobaca, Thioflavicoccus, and Thiorhodococcus.     

Induced expression of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ribonuclease III gene in transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. cv. SR1 tobacco plants

Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.     

RNase 1 genes from the family Sciuridae define a novel rodent ribonuclease cluster

The RNase A ribonucleases are a complex group of functionally diverse secretory proteins with conserved enzymatic activity. We have identified novel RNase 1 genes from four species of squirrel (order Rodentia, family Sciuridae). Squirrel RNase 1 genes encode typical RNase A ribonucleases, each with eight cysteines, a conserved CKXXNTF signature motif, and a canonical His¹²-Lys⁴¹-His¹¹⁹ catalytic triad. Two alleles encode Callosciurus prevostii RNase 1, which include a Ser¹⁸?Pro, analogous to the sequence polymorphisms found among the RNase 1 duplications in the genome of Rattus exulans. Interestingly, although the squirrel RNase 1 genes are closely related to one another (77–95% amino acid sequence identity), the cluster as a whole is distinct and divergent from the clusters including RNase 1 genes from other rodent species. We examined the specific sites at which Sciuridae RNase 1s diverge from Muridae/Cricetidae RNase 1s and determined that the divergent sites are located on the external surface, with complete sparing of the catalytic crevice. The full significance of these findings awaits a more complete understanding of biological role of mammalian RNase 1s.     

Production and characterization of fungal <Emphasis Type="Italic">β</Emphasis>-glucosidase and bacterial cellulases by tobacco chloroplast transformation

The high capacity of the chloroplast genome to integrate and express transgenes at high levels makes transplastomic technology a good option for overexpressing proteins of interest. This report presents the stable expression of β-glucosidase (bgl1 gene) from Aspergillus niger and two cellulases (celA and celB genes) from Thermotoga neapolitana into the chloroplast genome of tobacco. The pES6, pHM4, pHM5 and pHM6 vectors were derived from the pES4 plasmid containing bgl1, celA-celB, celA and celB synthetic genes, respectively. All of the genes were flanked by a synthetic rrn16 promoter and the 3′UTR from rbcL gene. The integration of the genes into intergenic regions rrn16 and 3′rps12 of the inverted repeats was confirmed by Southern blot analysis. Stable expression and processing of monocistronic mRNA were confirmed by Northern blot analysis, and protein functionality was analysed via enzymatic activity assay. The recombinant enzymes exhibited high enzymatic activity at pH 5 (β-glucosidase: 30.45 U mg^?1 of TSP, celA-celB 58 U mg^?1 of TSP, celA 49.10 U mg^?1 of TSP and celB 48.72 U mg^?1 of TSP). In addition, β-glucosidase exhibited high activity at 40 °C, whereas cellulases type A (celA) and type B (celB) showed high activity at 65 °C. NtpES6, NtpHM5 and NtpHM6 plants showed a similar phenotype compared with the wild type plants; however, NtpHM4 plants presented an abnormal phenotype with variegated leaves. This study, demonstrated that hydrolytic genes such as bgl1, celA and celB could be integrated and expressed correctly in the chloroplast genome. This work provides new information on methods and strategies for the expression of hydrolytic enzymes that are potentially useful for biotechnological applications using transplastomic plants.     

RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity

RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.     

Distribution and characteristics of <Emphasis Type="Italic">Bacillus</Emphasis> bacteria associated with hydrobionts and the waters of the Peter the Great Bay,Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

RraAS1 inhibits the ribonucleolytic activity of RNase ES by interacting with its catalytic domain in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.     

Development of RNA aptamer that inhibits methyltransferase activity of dengue virus

Objectives

To develop an RNA aptamer specific for the methyltransferase (MTase) of dengue virus (DENV) which is essential for viral genome replication and translation acting directly on N-7 and 2′-O-methylation of the type-I cap structure of the viral RNA.

Results

We identified 2′-fluoro-modified RNA aptamers that can specifically bind DENV serotype 2 (DENV2) MTase using systematic evolution of ligands by exponential enrichment technology. We truncated the chosen aptamer into a 45-mer RNA sequence that can bind DENV2 MTase with K _d  ~ 28 nM and inhibit N-7 methylation activity of the protein. Moreover, the 45-mer truncated aptamer could not only bind with an K _d  ~ 15.6 nM but also inhibit methylation activity of DENV serotype 3 (DENV3) MTase. The 45-mer aptamer competitively impeded binding of both DENV2 and DENV3 genomic RNA to MTase of each serotype.

Conclusion

The selected 45-mer truncated RNA aptamer specifically and avidly bound DENV MTase and competitively inhibited its methylation activity, and thus could be useful for the development of anti-DENV agents.

     

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of β-deletion splice variant was determined during EndoG overexpression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei as well as after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.     

Binase-Induced Changes of Tumor Cell Membranes

Exogenous ribonucleases of Bacilli can selectively induce apoptosis of malignant cells. The ability of Bacillus pumilus ribonuclease, binase, to induce processes leading to a dynamic disruption of the integrity of A549 human pulmonary adenocarcinoma cell membranes was analyzed. The influence of different enzyme concentrations on the state of the cytoplasmic membrane of cells and mitochondrial membranes was characterized. Using the methods of flow cytofluorometry and fluorescence microscopy, it has been established that binase leads to disruption in normal functioning of both types of membranes, with mitochondrial membranes affected first. The study made it possible to identify and visualize the effects of binase on the membrane structures of target cells and to confirm that bacterial RNase induces apoptosis of target cells mainly through the “internal” (mitochondrial) pathway.     

Changes in the Functional Activity of Phi11 Cro Protein is Mediated by Various Ions

Phi11, a temperate bacteriophage of Staphylococcus aureus, has been found to harbor a cro repressor gene which facilitates Phi11 to adopt the lytic mode of development. The Cro protein has been found to bind very specifically to a 15-bp operator DNA, located in the Phi11 cI–cro intergenic region [1]. To investigate the effects exerted by different ions upon the interaction between Cro and its cognate operator DNA, we have employed gel shift assays as well as circular dichroism spectral analysis. In this communication, we have shown that NH₄ ⁺ and acetate^? ions better facilitated the binding of Cro with its cognate operator as compared to Na⁺, K⁺ and Li⁺. Interestingly, Mg²⁺, carbonate^2? and Citrate^3? have an inhibitory effect upon the binding. The effect of the said ions upon the structure of Cro was also investigated by circular dichroism and it was found that other than Citrate^3? ions, none of the other ions destabilised the protein. On the other hand, Mg²⁺ and carbonate^2? ions maintained the structure of the protein but severely hampered its functional activity. Citrate^3? ions severely unfolded Cro and also inhibited its function. Considering all the data, NH₄ ⁺ and acetate^? ions appeared to be more suitable in maintaining the biological activity of Cro.