Dynamic guide-target interactions contribute to sequential 2'-O-methylation by a unique archaeal dual guide box C/D sRNP

Assembly and guide-target interaction of an archaeal box C/D-guide sRNP was investigated under various conditions by analyzing the lead (II)-induced cleavage of the guide RNA. Guide and target RNAs derived from Haloferax volcanii pre-tRNA(Trp) were used with recombinant Methanocaldococcus jannaschii core proteins in the reactions. Core protein L7Ae binds differentially to C/D and C'/D' motifs of the guide RNA, and interchanging the two motifs relative to the termini of the guide RNA did not affect L7Ae binding or sRNA function. L7Ae binding to the guide RNA exposes its D'-guide sequence first followed by the D guide. These exposures are reduced when aNop5p and aFib proteins are added. The exposed guide sequences did not pair with the target sequences in the presence of L7Ae alone. The D-guide sequence could pair with the target in the presence of L7Ae and aNop5p, suggesting a role of aNop5p in target recruitment and rearrangement of sRNA structure. aFib binding further stabilizes this pairing. After box C/D-guided modification, target-guide pairing at the D-guide sequence is disrupted, suggesting that each round of methylation may require some conformational change or reassembly of the RNP. Asymmetric RNPs containing only one L7Ae at either of the two box motifs can be assembled, but a functional RNP requires L7Ae at the box C/D motif. This arrangement resembles the asymmetric eukaryal snoRNP. Observations of initial D-guide-target pairing and the functional requirement for L7Ae at the box C/D motif are consistent with our previous report of the sequential 2'-O-methylations of the target RNA.     

Structurally conserved Nop56/58 N-terminal domain facilitates archaeal box C/D ribonucleoprotein-guided methyltransferase activity

Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.     

Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2'-O-methylation of the 3'-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.     

Alternative conformations of the archaeal Nop56/58-fibrillarin complex imply flexibility in box C/D RNPs

The Nop56/58-fibrillarin heterocomplex is a core protein complex of the box C/D ribonucleoprotein particles that modify and process ribosomal RNAs. The previous crystal structure of the Archaeoglobus fulgidus complex revealed a symmetric dimer of two Nop56/58-fibrillarin complexes linked by the coiled-coil domains of the Nop56/68 proteins. However, because the A. fulgidus Nop56/58 protein lacks some domains found in most other species, it was thought that the bipartite architecture of the heterocomplex was not likely a general phenomenon. Here we report the crystal structure of the Nop56/58-fibrillarin complex bound with methylation cofactor, S-adenosyl-L-methionine from Pyrococcus furiosus, at 2.7 A. The new complex confirms the generality of the previously observed bipartite arrangement. In addition however, the conformation of Nop56/58 in the new structure differs substantially from that in the earlier structure. The distinct conformations of Nop56/58 suggest potential flexibility in Nop56/58. Computational normal mode analysis supports this view. Importantly, fibrillarin is repositioned within the two complexes. We propose that hinge motion within Nop56/58 has important implications for the possibility of simultaneously positioning two catalytic sites at the two target sites of a bipartite box C/D guide RNA.     

The Nop5-L7A-fibrillarin RNP complex and a novel box C/D containing sRNA of Halobacterium salinarum NRC-1

RNA 2′O-methylation is a frequent modification of rRNA and tRNA and supposed to influence RNA folding and stability. Ribonucleoprotein (RNP) complexes, containing the proteins Nop5, L7A, fibrillarin, and a box C/D sRNA, are guided for 2′O-methylation by interactions of their RNA component with their target RNA. In vitro complex assembly was analyzed for several thermophilic Archaea but in vivo studies are rare, even unavailable for halophilic Archaea. To analyze the putative box C/D RNP complex in the extremely halophilic Halobacterium salinarum NRC-1 we performed pull-down analysis and identified the proteins Nop5, L7A, and fibrillarin and the tRNA^Trp intron, as a typical box C/D sRNA of this RNP complex in vivo. We show for the first time a ribonucleolytic activity of the purified RNP complex proteins, as well as for the RNP complex containing pull-down fractions. Furthermore, we identified a novel RNA (OE4630R-3′sRNA) as part of the complex, containing the typical boxes C/D and C′/D′ sequence motifs and being twice as abundant as the tRNA^Trp intron.     

Location of 2(')-O-methyl nucleotides in 26S rRNA and methylation guide snoRNAs in Caenorhabditis elegans

Many nucleotides in rRNAs are modified. We devised a method to locate 2(')-O-methyl nucleotide residues using a conventional DNA sequencer. We found 38 2(')-O-methyl nucleotides in the 26S rRNA of Caenorhabditis elegans using this method. Fourteen of the 38 residues are conserved in both human and yeast rRNAs and 14 residues are conserved in either human or yeast rRNA. The remaining 10 nucleotides are uniquely methylated in C. elegans 26S rRNA. We searched the C. elegans genomic sequence for small nucleolar RNAs (snoRNAs), which guide the methylation of ribose residues, and predicted 18 snoRNA sequences that are expected to guide the methylation of some of these nucleotide residues.     

Synthesis and properties of novel 2'-O-alkoxymethyl-modified nucleic acids

Novel 2'-O-modified oligoribonucleotides with alkoxymethyl skeletons were synthesized, and their ability to hybridize complementary nucleic acids and their nuclease resistance were analyzed. The hybridization ability was improved by introducing electron-withdrawing groups and the increases in melting temperature (T(m) value) was particularly high for chlorine-substituted compounds. Nuclease resistance of these 2'-O-alkoxymethylated oligomers was lower than expected, but cyano substitution resulted in a higher nuclease resistance than 2'-O-methylation.     

Exploration of pairing constraints identifies a 9 base-pair core within box C/D snoRNA-rRNA duplexes

2'-O-ribose methylation of eukaryotic ribosomal RNAs is guided by RNA duplexes consisting of rRNA and box C/D small nucleolar (sno)RNA sequences, the methylated sites invariably mapping five positions apart from the D box. Here we have analyzed the RNA duplex pairing constraints by investigating the features of 415 duplexes from the fungus, plant and animal kingdoms, and the evolution of those duplexes within the 124 sets they group into. The D-box upstream 1st and >or=15th positions consist of Watson-Crick base-pairs, G:U base-pairs and mismatched bases with ratios close to random assortments; these positions display single base differences in >60% of the RNA duplex sets. The D-box upstream 2nd to 11th positions have >90% Watson-Crick base-pairs; they display single base mutations with a U-shaped distribution of lower values of 0% and 1.6% at the methylated site 5th and 4th positions, and double compensatory mutations leading to new Watson-Crick base-pairs with an inverted U-shaped distribution of higher values at the 8th to 11th positions. Half of the single mutations at the 3rd to 11th positions resulted in G:U base-pairing, mainly through A-->G mutations in the rRNA strands and C-->T mutations in the snoRNA strands. Double compensatory mutations at the 3rd to 11th positions are extremely frequent, representing 36% of all mutations; they frequently arose from an A-->G mutation in the rRNA strands followed by a T-->C mutation in the snoRNA strands. Differences in the mutational pathways through which the rRNA and snoRNA strand evolved must be related to differences in the rRNA and snoRNA copy number and gene organization. Altogether these data identify the D-box upstream 3rd to 11th positions as box C/D snoRNA-rRNA duplex cores. The impact of the pairing constraints on the evolution of the 9 base-pair RNA duplex cores is discussed.     

A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs

We have identified a novel archaeal protein that apparently plays two distinct roles in ribosome metabolism. It is a polypeptide of about 18 kDa (termed Rbp18) that binds free cytosolic C/D box sRNAs in vivo and in vitro and behaves as a structural ribosomal protein, specifically a component of the 30S ribosomal subunit. As Rbp18 is selectively present in Crenarcheota and highly thermophilic Euryarchaeota, we propose that it serves to protect C/D box sRNAs from degradation and perhaps to stabilize thermophilic 30S subunits.     

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.     

Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping

Ribosomal RNAs contain numerous modifications at specific nucleotides. Despite their evolutionary conservation, the functional role of individual 2'-O-ribose methylations in rRNA is not known. A distinct family of small nucleolar RNAs, box C/D snoRNAs, guides the methylating complex to specific rRNA sites. Using a high-resolution phenotyping approach, we characterized 20 box C/D snoRNA gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intraribosomal antibiotics, inhibitors of specific cellular features, as well as general stressors. Ribosome-specific antibiotics generated phenotypes indicating different and long-ranging structural effects of rRNA methylations on the ribosome. For all studied box C/D snoRNA mutants we uncovered phenotypes to extraribosomal growth inhibitors, most frequently reflected in alteration in growth lag (adaptation time). A number of strains were highly pleiotropic and displayed a great number of sensitive phenotypes, e.g., deletion mutants of snR70 and snR71, which both have clear human homologues, and deletion mutants of snR65 and snR68. Our data indicate that individual rRNA ribose methylations can play either distinct or general roles in the workings of the ribosome.     

The C-terminal region of the exosome-associated protein Rrp47 is specifically required for box C/D small nucleolar RNA 3'-maturation

Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3'-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.     

Lysosome destabilization by cytosolic extracts, putative involvement of Ca(2+)/phospholipase C

Lysosomal disintegration is a crucial event for living cells, but mechanisms for the event are still unclear. In this study, we established that the cytosolic extracts could enhance lysosomal osmotic sensitivity and osmotically destabilize the lysosomes. The cytosol also caused the lysosomes to become more swollen in the hypotonic sucrose medium. The results indicate that the cytosol induced an osmotic shock to the lysosomes and an influx of water into the organelle. Since the effects of cytosol on the lysosomes could be abolished by O-tricyclo[5.2.1.0(2,6)]dec-9-yl dithiocarbonate potassium salt (D609), a specific inhibitor of cytosolic phospholipase C (PLC), the PLC might play an important role in the lysosomal osmotic destabilization. The activity of cytosolic PLC and the extent of enzyme latency loss of the cytosol-treated lysosomes exhibited a similar biphasic dependence on the cytosolic Ca2+ concentration. In addition, the cytosol did not osmotically destabilize the lysosomes until the cytosolic calcium ions rose above 100 nM. It suggests that the destabilization effect of cytosol on the lysosomes is Ca(2+)-dependent.     

Mutations in a sequence near the N-terminus of the small subunit alter the CO2/O2specificity factor for ribulose bisphosphate carboxylase/oxygenase

Seven unique substitutions have been introduced by site-directed mutagenesis into the first conserved region of the small subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans 6301. After expression of each large, altered-small subunit gene tandem in Escherichia coli, two substitutions in the small subunit tyr17asp17 (Y17D) and arg10gly10 (R10G) result in little or no carboxylase activity. For the latter substitution, no L₈S₈enzyme complex could be detected suggesting that this mutation prevents assembly. Mutant enzymes containing the following substitutions R11G, T14A, S16A, Y17D and P19A have CO₂/O₂specificity factors ( values) of 40, 35, 18, 39 and 44, respectively, compared with that of 44 for wild-type recombinant enzyme whereas P20A has full carboxylase activity and a value of 55.     

De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is an edible root vegetable crop that is cultivated worldwide and whose genome has been sequenced. Here we report the complete nucleotide sequence of the radish cultivar WK10039 chloroplast (cp) genome, along with a de novo assembly strategy using whole genome shotgun sequence reads obtained by next generation sequencing. The radish cp genome is 153,368 bp in length and has a typical quadripartite structure, composed of a pair of inverted repeat regions (26,217 bp each), a large single copy region (83,170 bp), and a small single copy region (17,764 bp). The radish cp genome contains 87 predicted protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence analysis revealed the presence of 91 simple sequence repeats (SSRs) in the radish cp genome.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.