Biomedical subjects
Akira Muto
Publications and source records attributed to Akira Muto.
Twelve novel C. elegans RNA candidates isolated by two-dimensional polyacrylamide gel electrophoresis.
C. elegans small RNAs (<50 nt) were separated by two-dimensional gel electrophoresis (2D-PAGE). cDNAs were prepared from the RNAs extracted from randomly chosen 2D-PAGE spots. Although many cDNA sequences corresponded to parts of known RNAs, twelve novel small RNA candidates were identified: eleven from 2D-PAGE spots of the mixed-stage worm RNA preparation and one from those of the embryonic RNA preparation. These are encoded in the intergenic regions, in the introns of protein-coding genes, in the anti-sense strand of protein-coding sequences and repetitive sequence regions of the genome. None of them showed a characteristic structure of miRNAs, suggesting that they are candidates of other or new classes of RNAs.
Forward genetic analysis of visual behavior in zebrafish.
The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.
Competition between trans-translation and termination or elongation of translation.
The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3'-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3'-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.
Interaction analysis between tmRNA and SmpB from Thermus thermophilus.
Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.
Functional analysis of factors involved in trans-translation.
trans-translation is a rescue system for stalled ribosomes to terminate the translation for recycling of ribosome and lead the incomplete protein on the stalled ribosome to degradation. In trans-translation, several proteins and RNAs are involved. tmRNA and SmpB are only factors known to be essential for trans-translation. EF-Tu and EF-G functions in translation and also are seemed to function in trans-translation. Ribosomal protein S1 is known to bind tmRNA. In this study, we cloned the gene of tmRNA from Thermus thermophilus and analyzed the functions of factors involved in trans-translation in vitro.
Participation of 3'-to-5' exoribonucleases in the turnover of Bacillus subtilis mRNA.
Four 3'-to-5' exoribonucleases have been identified in Bacillus subtilis: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5' ends were at the transcriptional start site and whose 3' ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of rpsO mRNA decay showed that decay intermediates accumulated as the result of a block to 3'-to-5' processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA trans translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.
Retinal network adaptation to bright light requires tyrosinase.
The visual system adjusts its sensitivity to a wide range of light intensities. We report here that mutation of the zebrafish sdy gene, which encodes tyrosinase, slows down the onset of adaptation to bright light. When fish larvae were challenged with periods of darkness during the day, the sdy mutants required nearly an hour to recover optokinetic behavior after return to bright light, whereas wild types recovered within minutes. This behavioral deficit was phenocopied in fully pigmented fish by inhibiting tyrosinase and thus does not depend on the absence of melanin pigment in sdy. Electroretinograms showed that the dark-adapted retinal network recovers sensitivity to a pulse of light more slowly in sdy mutants than in wild types. This failure is localized in the retinal neural network, postsynaptic to photoreceptors. We propose that retinal pigment epithelium (which normally expresses tyrosinase) secretes a modulatory factor, possibly L-DOPA, which regulates light adaptation in the retinal circuitry.
A novel GTPase activated by the small subunit of ribosome.
The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.
A minimum structure of aminoglycosides that causes an initiation shift of trans-translation.
Trans-translation is an unusual translation in which transfer-messenger RNA plays a dual function--as a tRNA and an mRNA--to relieve the stalled translation on the ribosome. It has been shown that paromomycin, a typical member of a 4,5-disubstituted class of aminoglycosides, causes a shift of the translation-resuming point on the tmRNA by -1 during trans-translation. To address the molecular basis of this novel effect, we examined the effects of various aminoglycosides that can bind around the A site of the small subunit of the ribosome on trans-translation in vitro. Tobramycin and gentamicin, belonging to the 4,6-disubstituted class of aminoglycosides having rings I and II similar to those in the 4,5-disubstituted class, possess similar effects. Neamine, which has only rings I and II, a common structure shared by 4,5- and 4,6-disubstituted classes of aminoglycosides, was sufficient to cause an initiation shift of trans-translation. In contrast, streptomycin or hygromycin B, lacking ring I, did not cause an initiation shift. The effect of each aminoglycoside on trans-translation coincides with that on conformational change in the A site of the small subunit of the ribosome revealed by recent structural studies: paromomycin, tobramycin and geneticin which is categorized into the gentamicin subclass, but not streptomycin and hygromycin B, flip out two conserved adenine bases at 1492 and 1493 from the A site helix. The pattern of initiation shifts by paromomycin fluctuates with variation of mutations introduced into a region upstream of the initiation point.
In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide.
Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.
Behavioral screening assays in zebrafish.
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Initiation-shift of trans-translation by aminoglycosides.
Trans-translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of several kinds of aminoglycoside antibiotics, on trans-translation in vitro. A chemical footprinting study revealed that paromomycins bind tmRNA in the tRNA domain and in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound in the tRNA domain inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA binding protein. It was also found that paromomycin causes a shift of the translation-resuming point on tmRNA by -1. The effect on initiation-shift was suppressed by a mutation at the paromomycin binding site in 16S rRNA, but not by mutations in tmRNA. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome. A minimal structure that causes initiation-shift was identified.
[Trans-translation: co-translation protein quality control system].
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Distribution of the MCS4 RNA genes in mycoplasmas belonging to the Mycoplasma mycoides cluster.
MCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum subsp. capricolum type strain California kid. This RNA has a sequence similarity to that of eukaryotic U6 snRNA. There are two genes encoding MCS4 RNA, designated mcs4a and mcs4b, in the genome. Homologous sequences of these genes were not found in databases of other bacterial sequences. We searched for MCS4 RNA and its genes in other bacteria by PCR and hybridization techniques. The results strongly suggested that this RNA exists only in a limited species of mycoplasmas belonging to the Mycoplasma mycoides cluster.
Various effects of paromomycin on tmRNA-directed trans-translation.
trans-Translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. The results of a chemical footprinting study indicated that paromomycin molecules bind tmRNA at G332/G333 in the tRNA domain and A316 in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound at G332/G333 inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA-binding protein. It was also found that paromomycin causes a shift of the translation resuming point on tmRNA by -1. The effect on initiation shift was canceled by a mutation at the paromomycin-binding site in 16 S rRNA but not by mutations in tmRNA. A high concentration of paromomycin inhibited trans-translation, whereas it enhanced the initiation-shifted trans-translation when SmpB was exogenously added or a mutation was introduced at 333. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.
Various effects of paromomycin on tmRNA-mediated trans-translation.
Trans-translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. A chemical footprinting study revealed that paromomycins bind tmRNA in the tRNA domain and in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound in the tRNA domain inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA binding protein. It was also found that paromomycin causes a shift of the translation-resuming point on tmRNA by -1. The effect on initiation-shift was canceled by a mutation at the paromomycin binding site in 16S rRNA, but not by mutations in tmRNA. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.
In vitro analysis of the initial steps of trans-translation.
tmRNA has a dual function both as tRNA and mRNA and facilitates trans-translation. We observed the tagging derived from tmRNA from Bacillus subtilis in vivo and in vitro. Our studies give progressive suggestions on the mechanism of trans-translation.