MicroRNA 210 as a biomarker for congestive heart failure.

MicroRNAs (miRNAs) are endogenous small RNAs that are 18-23 nucleotides long. Recently, plasma miRNAs were reported to be sensitive and specific biomarkers of various pathological conditions. In the present study, we focused on miR-210, which is known to be induced by hypoxia and might therefore be an excellent biomarker for congestive heart failure. Plasma miR-210 levels and expression levels in mononuclear cells and skeletal muscles were elevated in Dahl salt-sensitive rats with heart failure. We also assessed miR-210 expression in patients with heart failure. The miR-210 expression levels in the mononuclear cells of patients with NYHA III and IV heart failure according to the New York Heart Association (NYHA) functional classification system were significantly higher than those with NYHA II heart failure and controls. Although no significant correlation was observed between plasma brain natriuretic peptide (BNP) and plasma miR-210 levels in patients with NYHA II heart failure, patients with an improved BNP profile at the subsequent hospital visit were classified in a subgroup of patients with low plasma miR-210 levels. Plasma miR-210 levels may reflect a mismatch between the pump function of the heart and oxygen demand in the peripheral tissues, and be a new biomarker for chronic heart failure in addition to plasma BNP concentrations.

Pex11α deficiency impairs peroxisome elongation and division and contributes to nonalcoholic fatty liver in mice.

Hepatic triglyceride (TG) accumulation is considered to be a prerequisite for developing nonalcoholic fatty liver (NAFL). Peroxisomes have many important functions in lipid metabolism, including fatty acid ß-oxidization. However, the pathogenic link between NAFL and peroxisome biogenesis remains unclear. To examine the molecular and physiological functions of the Pex11α gene, we disrupted this gene in mice. Body weights and hepatic TG concentrations in Pex11α(-/-) mice were significantly higher than those in wild-type (WT) mice fed a normal or a high-fat diet. Hepatic TG concentrations in fasted Pex11α(-/-) mice were significantly higher than those in fasted WT mice. Plasma TG levels increased at lower rates in Pex11α(-/-) mice than in WT mice after treatment with the lipoprotein lipase inhibitor tyloxapol. The number of peroxisomes was lower in the livers of Pex11α(-/-) mice than in those of WT mice. Ultrastructural analysis showed that small and regular spherically shaped peroxisomes were more prevalent in Pex11α(-/-) mice fed normal chow supplemented without or with fenofibrate. We observed a significantly higher ratio of empty peroxisomes containing only PMP70, a peroxisome membrane protein, but not catalase, a peroxisome matrix protein, in Pex11α(-/-) mice. The mRNA expression levels of peroxisomal fatty acid oxidation-related genes (ATP-binding cassette, subfamily D, member 2, and acyl-CoA thioesterase 3) were significantly higher in WT mice than those in Pex11α(-/-) mice under fed conditions. Our results demonstrate that Pex11α deficiency impairs peroxisome elongation and abundance and peroxisomal fatty acid oxidation, which contributes to increased lipid accumulation in the liver.

P2X(7) receptor antagonism attenuates the hypertension and renal injury in Dahl salt-sensitive rats.

The P2X(7) receptor is a ligand-gated ion channel activated by extracellular ATP, and a common genetic variation in the P2X(7) gene significantly affects blood pressure. P2X(7) receptor expression is associated with renal injury and some inflammatory diseases. Brilliant blue G (BBG) is a selective rat P2X(7) receptor antagonist. In this study, to test whether BBG has protective effects on salt-sensitive hypertension and renal injury, Dahl salt-sensitive (DS) rats fed an 8% NaCl diet were i.p. injected with BBG (50 mg kg(-1) per day) for 4 weeks. We also tested another P2X(7) receptor antagonist, namely A-438079 (100 mg kg(-1) per day), for 7 days. We found that P2X(7) antagonism markedly attenuated salt-sensitive hypertension, urinary protein or albumin excretion, renal interstitial fibrosis and macrophage and T-cell infiltration in the DS rats, and significantly improved creatinine clearance. In an in vitro experiment using macrophages, we showed that lipopolysaccharide (LPS)-primed macrophages from the DS rats released more interleukin-1 beta in response to BzATP, a P2X(7) receptor agonist, than the macrophages from Lewis rats, possibly due to higher P2X(7) expression in the DS rats. In conclusion, in vivo blockade of P2X(7) receptors attenuated salt-sensitive hypertension and renal injury in the DS rats. Thus, P2X(7) appears to be responsible for a vicious cycle of salt-sensitive hypertension and renal injury in the DS rats, through higher expression in the immune cells. Furthermore, P2X(7) antagonists can prevent the development of salt-sensitive hypertension and renal injury, thus confirming that the P2X(7) receptor is an important therapeutic target.

Plasma miR-208 as a biomarker of myocardial injury.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small RNAs of 21-25 nucleotides that can pair with sites in 3' untranslated regions in mRNAs of protein-coding genes to downregulate their expression. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. We assessed the hypothesis that miRNAs may leak into the circulating blood from injured cells and thereby serve as biomarkers for identifying the injured cell type. METHODS: We used isoproterenol-induced myocardial injury in rats as a model and miRNA array analyses to identify candidate miRNAs specifically produced in the ventricles of the heart. Individual miRNA concentrations were measured by real-time reverse-transcription PCR. Plasma cardiac troponin I (cTnI) concentrations were measured with an ELISA. RESULTS: Array analyses revealed miR-208 to be produced exclusively in the heart, and we selected this miRNA as a possible biomarker of myocardial injury. Plasma concentrations of miR-208 increased significantly (P < 0.0001) after isoproterenol-induced myocardial injury and showed a similar time course to the concentration of cTnI, a classic biomarker of myocardial injury. CONCLUSIONS: The plasma concentration of miR-208 may be a useful indicator of myocardial injury. Our results suggest that profiling of circulating miRNAs may help identify promising biomarkers of various pathologic conditions.

Targeted deletion of miR-182, an abundant retinal microRNA.

PURPOSE: MicroRNA-182 (miR-182) is expressed abundantly in the mammalian retina and is therefore thought to perform important roles for the retinal development and the function. To test this hypothesis, we generated miR-182 knockout mice. METHODS: northern blotting was performed to confirm the robust expression of miR-182 in the eye. The precursor sequence of miR-182 was replaced by the neomycin resistance gene under the control of the phosphoglycerate kinase 1 promoter in a targeting construct. The targeting vector was linearized and transfected into embryonic stem (ES) cells. Recombinant ES clones were selected and injected into blastocysts to generate male chimeras. Heterozygous and homozygous mice were obtained after five generations of backcrossing and were confirmed using genotyping and northern blotting. RESULTS: Heterozygous (+/-) and homozygous (-/-) knockout mice were morphologically normal, viable, and fertile. Immunohistochemical analysis of the miR-182-deficient retinas did not reveal any apparent structural abnormalities in the retinas. Consistently, global expression profiling using a repeated microarray did not identify significant fluctuations for potential target genes. CONCLUSIONS: We successfully generated miR-182 knockout mice and characterized the resulting miR-182-depleted retina. This is the first report describing the targeted deletion of a single miRNA that is highly expressed in the retina. The absence of significant transcriptional and phenotypic changes in miR-182-depleted retinas suggests that miR-182 is not a major determinant of retinal development or delamination. Further studies are required to elucidate any functional changes in the retina.

The role of GTP in transient splitting of 70S ribosomes by RRF (ribosome recycling factor) and EF-G (elongation factor G).

Ribosome recycling factor (RRF), elongation factor G (EF-G) and GTP split 70S ribosomes into subunits. Here, we demonstrated that the splitting was transient and the exhaustion of GTP resulted in re-association of the split subunits into 70S ribosomes unless IF3 (initiation factor 3) was present. However, the splitting was observed with sucrose density gradient centrifugation (SDGC) without IF3 if RRF, EF-G and GTP were present in the SDGC buffer. The splitting of 70S ribosomes causes the decrease of light scattering by ribosomes. Kinetic constants obtained from the light scattering studies are sufficient to account for the splitting of 70S ribosomes by RRF and EF-G/GTP during the lag phase for activation of ribosomes for the log phase. As the amount of 70S ribosomes increased, more RRF, EF-G and GTP were necessary to split 70S ribosomes. In the presence of a physiological amount of polyamines, GTP and factors, even 0.6 microM 70S ribosomes (12 times higher than the 70S ribosomes for routine assay) were split. Spermidine (2 mM) completely inhibited anti-association activity of IF3, and the RRF/EF-G/GTP-dependent splitting of 70S ribosomes.

Structural Insights into ribosome recycling factor interactions with the 70S ribosome.

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 A, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.

Minor contribution of an internal ribosome entry site in the 5'-UTR of ornithine decarboxylase mRNA on its translation.

The mechanism of synthesis of ornithine decarboxylase (ODC) at the level of translation was studied using cell culture and cell-free systems. Synthesis of firefly luciferase (Fluc) from the second open reading frame (ORF) in a bicistronic construct transfected into FM3A and HeLa cells was enhanced by the presence of the 5'-untranslated region (5'-UTR) of ODC mRNA between the two ORFs. However, cotransfection of the gene encoding 2A protease inhibited the synthesis of Fluc. Synthesis of Fluc from the second cistron in the bicistronic mRNA in a cell-free system was not affected significantly by the 5'-UTR of ODC mRNA. Synthesis of ODC from ODC mRNA in a cell-free system was inhibited by 2A protease and cap analogue (m7GpppG). Rapamycin inhibited ODC synthesis by 40-50% at both the G1/S boundary and the G2/M phase. These results indicate that an IRES in the 5'-UTR of ODC mRNA does not function effectively.

Structural basis for aminoglycoside inhibition of bacterial ribosome recycling.

Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.

Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP.

Ribosomes must dissociate into subunits in order to begin protein biosynthesis. The enzymes that catalyze this fundamental process in eukaryotes remained unknown. Here, we demonstrate that eukaryotic translocase, eEF2, which catalyzes peptide elongation in the presence of GTP, dissociates yeast 80S ribosomes into subunits in the presence of ATP but not GTP or other nucleoside triphosphates. Dissociation was detected by light scattering or ultracentrifugation after the split subunits were stabilized. ATP was hydrolyzed during the eEF2-dependent dissociation, while a non-hydrolyzable analog of ATP was inactive in ribosome splitting by eEF2. GTP inhibited not only ATP hydrolysis but also dissociation. Sordarin, a fungal eEF2 inhibitor, averted the splitting but stimulated ATP hydrolysis. Another elongation inhibitor, cycloheximide, also prevented eEF2/ATP-dependent splitting, while the inhibitory effect of fusidic acid on the splitting was nominal. Upon dissociation of the 80S ribosome, eEF2 was found on the subunits. We propose that the dissociation activity of eEF2/ATP plays a role in mobilizing 80S ribosomes for protein synthesis during the shift up of physiological conditions.

Inhibition of antiassociation activity of translation initiation factor 3 by paromomycin.

The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg(2+). Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.

The ribosome-recycling step: consensus or controversy?

Ribosome recycling, the last step in translation, is now accepted as an essential process for prokaryotes. In 2005, three laboratories showed that ribosome-recycling factor (RRF) and elongation factor G (EF-G) cause dissociation of ribosomes into subunits, solving the long-standing problem of how this essential step of translation occurs. However, there remains ongoing controversy regarding the other actions of RRF and EF-G during ribosome recycling. We propose that the available data are consistent with the notion that RRF and EF-G not only split ribosomes into subunits but also participate directly in the release of deacylated tRNA and mRNA for the next round of translation.

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits.

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.

In vivo effect of inactivation of ribosome recycling factor - fate of ribosomes after unscheduled translation downstream of open reading frame.

The post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Without RRF, the ribosome is not released from mRNA at the termination codon and reinitiates translation downstream. This is called unscheduled translation. Here, we show that at the non-permissive temperature of a temperature-sensitive RRF strain, RRF is lost quickly, and some ribosomes reach the 3' end of mRNA. However, instead of accumulating at the 3' end of mRNA, ribosomes are released as monosomes. Some ribosomes are transferred to transfer-messenger RNA from the 3' end of mRNA. The monosomes thus produced are able to translate synthetic homopolymer but not natural mRNA with leader and canonical initiation signal. The pellet containing ribosomes appears to be responsible for rapid but reversible inhibition of most but not all of protein synthesis in vivo closely followed by decrease of cellular RNA and DNA synthesis.

Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs.

It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits. In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon. In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs. In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes. Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases. These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications.

After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosome-recycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by approximately 13 A) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA.

Temperature-sensitive mutation in yeast mitochondrial ribosome recycling factor (RRF).

The yeast protein Rrf1p encoded by the FIL1 nuclear gene bears significant sequence similarity to Escherichia coli ribosome recycling factor (RRF). Here, we call FIL1 Ribosome Recycling Factor of yeast, RRF1. Its gene product, Rrf1p, was localized in mitochondria. Deletion of RRF1 leads to a respiratory incompetent phenotype and to instability of the mitochondrial genome (conversion to rho(-)/rho(0) cytoplasmic petites). Yeast with intact mitochondria and with deleted genomic RRF1 that harbors a plasmid carrying RRF1 was prepared from spores of heterozygous diploid yeast. Such yeast with a mutated allele of RRF1, rrf1-L209P, grew on a non-fermentable carbon source at 30 but not at 36 degrees C, where mitochondrial but not total protein synthesis was 90% inhibited. We propose that Rrf1p is essential for mitochondrial protein synthesis and acts as a RRF in mitochondria.

Binding of ribosome recycling factor to ribosomes, comparison with tRNA.

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.

Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic.

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.