Isolation of eight novel Caenorhabditis elegans small RNAs.

Eight novel small RNAs that were encoded in the regions corresponding to the introns of protein-coding genes were isolated from Caenorhabditis elegans. Seven of them showed a typical snoRNA secondary structure: one C/D snoRNA and six H/ACA snoRNAs. The remaining one RNA did not show any homology to other RNAs in a database. Four of the seven isolated snoRNAs could form base pairings with parts of rRNAs, suggesting that they are potential pseudouridilation sites and methylation sites. The results of our study suggest that there are more as-yet-unidentified small ncRNAs of which genes are located in the intron regions of protein-coding genes in C. elegans.

[Cognitive function and brain atrophy in elderly type 2 diabetic patients in comparison with non-diabetic elderly subjects].

Previous studies have suggested that type 2 diabetic mellitus could lead to learning and memory deficits. We studied cognitive function tests and brain computed tomography (CT) findings in elderly subjects with drug-treated type 2 diabetic patients (n = 9), diet-treated type 2 diabetic patients (n = 8) and nondiabetic subjects (CR, n = 21). A battery of cognitive function tests (Cog-T; WAIS-R's digit span test and symbol test, Stroop Test, ADAS's verbal memory test, and MMSE) was carried out on two occasions, separated by at least 6 months. Brain CT was analyzed by the following 5 variables; 1) Evan's Ratio, 2) Inverse Cella Media Index, 3) maximum width of the third ventricle, 4) maximum width of temporal horn tips on both sides and 5) maximum width of the Sylvian fissure at the insula, bilaterally. The scores of Cog-T did not differ significantly between the groups. On brain CT measurements, maximum width of the temporal horn tips on right side were significantly different in the three groups (ANOVA, P = 0.035). The drug-treated diabetics subjects had wider temporal horn tips on the right side than did the diet treated diabetics and nondiabetic subjects (Fisher's post hoc test, P = 0.030, P = 0.016).

Requirement of transfer-messenger RNA for the growth of Bacillus subtilis under stresses.

BACKGROUND: Bacterial transfer-messenger RNA (tmRNA, 10Sa RNA) is involved in a trans-translation reaction which contributes to the degradation of incompletely synthesized peptides and to the recycling of stalled ribosomes. However, its physiological role in the cell remains elusive. In this study, an efficient system for controlling the expression of the gene for tmRNA (ssrA), as well as a tmRNA gene-defective strain (ssrA:cat), were constructed in Bacillus subtilis. The effects of tmRNA on the growth of the cells were investigated under various physiological culture conditions using these strains. RESULTS: The cells were viable in the absence of ssrA expression under the usual culture conditions. However, the growth rate of cells without tmRNA expression, relative to that of the expressed cells, decreased with elevating temperature (> 45 degrees C), and at 52 degrees C, the highest temperature for growth of the wild-type, cells grew depending on the expression level of tmRNA. Furthermore, the transcription level of the ssrA from the authentic promoter at a high temperature (51 degrees C) was about 10-fold higher than that at a lower temperature (37 degrees C). tmRNA-dependent growth and an increase in tmRNA amount were also observed in cells under other stresses, such as high concentrations of ethanol or cadmium chloride. It is also shown that alanylated tmRNA rather than tmRNA-mediated proteolysis is required for growth at high temperature. CONCLUSION: The expression of tmRNA gene (ssrA) is required for the efficient growth of B. subtilis under several strong stresses. The transcription of ssrA increases under several stressful conditions, suggesting that it is a stress-response gene. Alanyl-tmRNA, probably via its ability of recycling stalled ribosomes via trans-translation, is involved in the stress tolerance of bacteria.

A bacterial RNA that functions as both a tRNA and an mRNA.

Bacterial tmRNA (transfer-messenger RNA, also known as 10Sa RNA) contains a tRNA-like structure in the 5'- and 3'-end sequences and an internal reading frame encoding a 'tag' peptide. The dual function of this molecule as both a tRNA and an mRNA facilitates a trans-translation reaction, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The result is a chimeric protein with the tag peptide attached to the C-terminus of the truncated peptide.

In vitro trans translation mediated by alanine-charged 10Sa RNA.

10Sa RNA is a bacterial small stable RNA, in which the 5' and 3'-terminal sequences can be folded into a tRNA-like secondary structure which can be aminoacylated with alanine. It was found that Escherichia coli 10Sa RNA facilitated the incorporation of alanine, tyrosine, aspartic acid and glutamic acid, but not valine, isoleucine, serine or arginine, into the growing polypeptide in vitro, depending on poly (U)-directed poly-phenylalanine synthesis. This result indicates that 10Sa RNA functions as an mRNA for the tag-peptide which has been found to be attached to the C termini of truncated polypeptides synthesized in vivo. Aminoacylation with alanine was required for tag-specific amino acid incorporation and for efficient association of 10Sa RNA with the ribosome, indicating that 10Sa RNA also functions as an alanine tRNA in the tag-peptide synthesis. The dual function of 10Sa RNA both as an mRNA and as a tRNA in vitro strongly supports the trans translation hypothesis.

Escherichia coli tmRNA (10Sa RNA) in trans-translation.

Here we show that Escherichia coli tmRNA (10Sa RNA) has a dual function both as an mRNA and as a tRNA in vitro. The function as a tRNA is prerequisite for the function as an mRNA. These observations strongly support the trans-translation hypothesis.

Purification of a Mycoplasma capricolum MCS4 RNA binding protein and cloning its gene.

MCS4 RNA (125 nt in length) is one of the small stable RNAs found in Mycoplasma capricolum cells. Gel shift assay was performed with the 5' end-32P-labeled MCS4 RNA and the S100 fraction from M. capricolum to identify the RNA binding proteins. Several bands were detected above free MCS4 RNA, indicating that the RNA formed complexes with proteins and/or RNAs. One of the proteins which specifically binds to MCS4 RNA was purified. The amino acid sequence of the N-terminus revealed 60 to 80% identity to those of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from various sources.

Probing the secondary structure of MCS4 RNA of Mycoplasma capricolum.

MCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum. Its function is unknown. The conformation of MCS4 RNA (125 nucleotides in length) in solution was investigated using chemical and enzymatic probes. Single and double-stranded regions were estimated by means of diethyl pyrocarbonate (DEPC) and by dimethyl sulfate (DMS) modifications. Ribonuclease V1 was also used to identify paired or stacked nucleotides. Based on these data, a secondary structure model for MCS4 RNA containing four stem structures was constructed.

Interaction of 10Sa RNA with ribosomes in Escherichia coli.

10Sa RNA is a bacterial small stable RNA, in which the 5'- and 3'-end sequences are folded into a tRNA-like structure. The RNA accepts alanine in vitro, and interacts with 70S ribosomes in the cells. In this study, we examined the ribosome-binding properties of Escherichia coli 10Sa RNA in vivo, and found that the aminoacylation ability of 10Sa RNA with alanine is necessary for the binding to 70S ribosomes. 10Sa RNA was also found to bind only to 70S monosomes and not to polysomes. Recently, E. coli 10Sa RNA was suggested to be used as mRNA for tag peptides, which were found to attach to the C-termini of truncated peptides synthesized in vivo. The present results are consistent with the 'trans-translation' model, which has been proposed for tag-peptide synthesis.

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Structure and function of 10Sa RNA: trans-translation system.

10Sa RNA is a small stable bacterial RNA in which the 5'- and 3'-end sequences are folded into a tRNA-like structure. The RNA is aminoacylatable with alanine in vitro, and it interacts with 70S ribosomes in the cell. Recently, Escherichia coli 10Sa RNA has been shown to contain the sequence-encoding tag-peptides, which are found to attach to the C-termini of truncated peptides synthesized in vivo. We have found that the E coli 10Sa RNA stimulates incorporation of the tag-specific amino acids into proteins depending on the poly(U)-directed poly-phenylalanine synthesis in the in vitro translation system. Our finding supports the 'trans-translation' model proposed for the tag synthesis, in which alanyl-10Sa RNA enters the ribosome when translation stops at the 3'-end of the truncated mRNA lacking a stop codon, and translation of the tag-peptide occurs by switching the template from mRNA to 10Sa RNA. In this unique reaction, 10Sa RNA acts both as a tRNA and as an mRNA.

Novel small stable RNAs of Mycoplasma capricolum.

Two stable RNA species and their genes have been isolated from Mycoplasma capricolum, and the nucleotide sequences have been determined by partial RNA sequencing and sequencing of the genes. The RNAs are 92 and 105 nucleotides in length, respectively. The two RNAs reveal no sequence similarity to any stable RNA so far reported, indicating that these are novel RNA species. The RNAs, designated MCS2 and MCS3 RNA, exist in small amounts in the soluble fraction of the cell extract.

RNase P RNA of Mycoplasma capricolum.

There are at least six small stable RNAs in Mycoplasma capricolum cells besides tRNAs and rRNAs. One of them, MCS5 RNA, is a homolog of RNase P RNA. The predicted secondary structure of this RNA is essentially the same as that of other eubacterial RNase P RNAs. MCS5 RNA is more similar to the RNase P RNA of B. Subtilis than to that of E. coli. This is consistent with previous conclusions that mycoplasmas are phylogenetically related to the low G + C Gram-positive bacterial group. The major substrates for MCS5 RNA must be the precursors of tRNAs. The precursor of MCS6 RNA, which is a homolog of the E. coli 10Sa RNA, may also be a substrate for the MCS5 RNA because this RNA has a tRNA-like structure at its 5' and 3' ends.

tRNA-like structures in 10Sa RNAs of Mycoplasma capricolum and Bacillus subtilis.

The stable RNAs, whose sequences are homologous to 10Sa RNA of Escherichia coli, have been isolated from Mycoplasma capricolum and Bacillus subtilis, both belonging to the Gram-positive bacterial group. The total nucleotide sequences of the RNAs have been determined by partial RNA sequencing and DNA sequencing of their genes. A comparison of the sequences, together with those of other bacterial 10Sa RNAs so far known, has shown that the 5'- and 3'-end sequences are well conserved among species, while the central parts reveal little homologies. Unexpectedly, the conserved 5'- and 3'-regions can be folded in a common tRNA-like structure containing an amino acid-acceptor stem and a T phi C-stem/loop. The 3'-terminal CCA sequence of B.subtilis 10Sa RNA is not encoded on the DNA, but is added after transcription. Furthermore, the RNA is aminoacylatable with alanine in vitro, and binds to the 70S ribosome in vivo.

A small RNA of Mycoplasma capricolum that resembles eukaryotic U6 small nuclear RNA.

Mycoplasma capricolum, a parasitic prokaryote, contains several small stable RNAs, besides rRNAs and tRNAs. One of them, designated MCS4 RNA (125 nucleotides in length), has been isolated and sequenced. This RNA is abundant in the cell, and is encoded by two genes. Unexpectedly, MCS4 RNA has been found to reveal extensive sequence similarity to eukaryotic U6 snRNAs. This finding suggests that MCS4 and U6 snRNAs are derived from a common ancestral RNA that has existed before the divergence of prokaryotes and eukaryotes.

Small stable RNAs in Mycoplasma capricolum.

Five small stable RNA species have been isolated from Mycoplasma capricolum. Together with the previously found RNA species, M.capricolum contains at least six small RNAs besides rRNAs and tRNAs. The sequences of these RNAs, designated MCS1 to MCS6, have been determined. MCS1 RNA is homologous to 4.5S RNA of E. coli, MCS5 to 10Sa RNA and MCS6 to M1 RNA (RNase P RNA). Unexpectedly, MCS4 RNA revealed an extensive sequence similarity to eukaryotic U6 snRNAs. MCS6, 10Sa RNA homolog, contains a tRNA-like structure in the 5'- and 3'-terminal sequences.