A novel cloverleaf structure found in mammalian mitochondrial tRNA(Ser) (UCN).

Bovine mitochondrial tRNA(Ser) (UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated the bovine liver mitochondrial tRNA(Ser) (UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5'-terminal nucleotide and enzymatic determination of the whole sequence of tRNA(Ser) (UCN) revealed that the tRNA started from the third nucleotide of the putative tRNA(Ser) (UCN) gene, which had formerly been supposed. Enzymatic probing of tRNA(Ser) (UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNA(Ser) (UCN) genes of mammals such as human, rat and mouse, but not for those of non-mammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria.     

精胺和Mg~（2＋）对tRNA~（Ile）圆二向色谱的影响

由于精胺（ｓｐｅｒｍｉｎｅ）能特异地刺激哺乳动物ｔＲＮＡ~（Ｉｌｅ）的氨基酰化,本文用纯化的牛肝ｔＲＮＡ~（Ｉｌｅ）观察了精胺和Ｍｇ（２＋）对ｔＲＮＡ~（Ｉｌｅ）ＣＤ光谱的影响。结果显示：Ｍｇ（２＋）可使牛肝ｔＲＮＡ~（Ｉｌｅ）ＣＤ光谱峰向短波方向偏移２ｎｍ,波峰为２６３ｎｍ,峰值被增大约１０％,ΔθＭｇ（２＋）＝２．３×１０３ｄｅｇ·ｃｍ２／ｄｍｏｌ;而精胺使牛肝ｔＲＮＡ~（Ｉｌｅ）ＣＤ光谱峰减少４０％,Δθｓｐｅｒｍｉｎｅ＝１×１０（－４）ｄｅｇ·ｃｍ２／ｄｍｏｌ;精胺和Ｍｇ（２＋）对肝ｔＲＮＡ~（Ｉｌｅ）－ＩｌｅＲＳ复合物或ＩｌｅＲＳ的ＣＤ光谱基本无影响。表明Ｍｇ（２＋）和精胺可影响牛肝ｔＲＮＡ~（Ｉｌｅ）的构象。实验同时以酵母ｔＲＮＡ（Ｐｈｅ）和Ｅ·ｃｏｌｉｔＲＮＡ~（Ｉｌｅ）作为对照。     

Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

1. tRNA isolated from non-lactating bovine mammary gland competitively inhibits the formation of aminoacyl-tRNA in the rat liver system. 2. Non-lactating bovine mammary gland tRNA and twice-pyrophosphorolysed rat liver tRNA are unable to accept amino acids in a reaction catalysed by aminoacyl-tRNA synthetases from either rat liver or bovine mammary gland. Deacylated rat liver tRNA can however be aminoacylated in the presence of either enzyme. 3. Bovine mammary gland tRNA lacks the terminal adenine nucleotide at the 3′-terminus amino acid acceptor end, which can be replaced by incubation in the presence of rat liver nucleotide-incorporating enzyme, ATP and CTP. 4. The enzymically modified bovine tRNA (tRNApCpCpA) can bind labelled amino acids to form aminoacyl-tRNA, which can then transfer its labelled amino acids to growing polypeptide chains on ribosomes. 5. Molecules of rat liver tRNA or bovine mammary gland tRNA that lack the terminal adenine nucleotide or the terminal cytosine and adenine nucleotides inhibit the aminoacylation of normal rat liver tRNA to varying degrees. tRNA molecules lacking the terminal −pCpCpA nucleotide sequence exhibit the major inhibitory effect. 6. The enzyme fraction from bovine mammary gland corresponding to that containing the nucleotide-incorporating enzyme in rat liver is unable to catalyse the incorporation of cytosine and adenine nucleotides in pyrophosphorolysed rat liver tRNA and deacylated bovine tRNA. This fraction also markedly inhibits the action of the rat liver nucleotide-incorporating enzyme.     

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.     

Identification of yeast tRNA Um(44) 2'-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA(Ser) species

A characteristic feature of tRNAs is the numerous modifications found throughout their sequences, which are highly conserved and often have important roles. Um(44) is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and unique to tRNA(Ser) in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNA(Ser) Um(44) 2'-O-methyltransferase. Trm44 was identified by screening a yeast genomic library of affinity purified proteins for activity and verified by showing that a trm44-delta strain lacks 2'-O-methyltransferase activity and has undetectable levels of Um(44) in its tRNA(Ser) and by showing that Trm44 purified from Escherichia coli 2'-O-methylates U(44) of tRNA(Ser) in vitro. Trm44 is conserved among metazoans and fungi, consistent with the conservation of Um(44) in eukaryotic tRNAs, but surprisingly, Trm44 is not found in plants. Although trm44-delta mutants have no detectable growth defect, TRM44 is required for survival at 33 degrees C in a tan1-delta mutant strain, which lacks ac(4)C12 in tRNA(Ser) and tRNA(Leu). At nonpermissive temperature, a trm44-delta tan1-delta mutant strain has reduced levels of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), but not other tRNA(Ser) or tRNA(Leu) species. The trm44-delta tan1-delta growth defect is suppressed by addition of multiple copies of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), directly implicating these tRNA(Ser) species in this phenotype. The reduction of specific tRNA(Ser) species in a trm44-delta tan1-delta mutant underscores the importance of tRNA modifications in sustaining tRNA levels and further emphasizes that tRNAs undergo quality control.     

Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes.

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.     

Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of (14)C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of (14)C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105 degrees for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.     

Trace 5-Methylaminomethyl-2-selenouridine in bovine tRNA and the selenouridine synthase activity in bovine liver

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. ⁷⁵Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T₂. The position of the ⁷⁵ Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from ⁷⁵Se-tRNA in E. coli.     

A study of the interaction between tRNASer and seryl-tRNA synthetase from bovine liver

Study by chemical modification of Ser, Arg, His residues and sulfhydryl groups on bovine seryl-tRNA synthetase showed that Ser residues appeared to be unnecessary for the recognition mechanism, but Arg and His residues were essential. It was considered that different sulfhydryl groups related with each recognition of tRNA and ATP. Poly-arginine inhibited the interaction between serine tRNA and SerRS. The CD spectra of a mixture of serine tRNA and poly-arginine indicated that higher-order structure of tRNA changed. Furthermore, the Km and Vmax values of bovine serine isoacceptor, yeast serine tRNA and E. coli serine tRNA for bovine SerRS examined and it was discussed the differences of those base sequences.     

The nucleotide sequence of arginine tRNACCG from bovine liver

The nuclotide sequence of arginine tRNA(CCG) from bovine liver was determined to be: pG-A-C-C-C-A-G-U-m(1)G-m(2)G-C-C-U-A-A-D-Gm-G-A-D-A-A-G-G-C-A-psi-C-A-G-C-Cm-U-C-C-G-m(1)G-A-G-C-U-G-G-G-G-A-D-U-G-psi-G-G-G-T-psi-C-G-m(1)A-G-U-C-C-C-A-U-C-U-G-G-G-U-C-G-C-C-A(OH). This arginine tRNA is 76 nucleotides in length with 13 modified bases and has an anticodon of CCG. The sequence of this molecule is substantially different from those of other arginine tRNAs sequenced to date and is the only arginine tRNA sequenced which would be expected to recognize the codon CGG.Images     

The nucleotide sequence of tyrosine tRNAQ* psi A from bovine liver

The nucleotide sequence of tyrosine tRNAQ* psi A from bovine liver was determined to be pC-C-U-U-C-m2G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3U-A-G-A -G-C-m22G-m22G -A-G-G-A-C-U-Q*-psi-A-m1G-A-psi m-C-C-U-U-A-G-m7G-D-m5C-G-C-U-G-G-T-psi-C-G-m1A -U-U-C-C-G-G-C-U-C-G-A-A-G-G-A-C-C-AOH. This tyrosine tRNA is 76 nucleotides in length, and contains two hypermodified nucleosides--3 -3(3-amino-3-carboxylpropyl)uridine (acp3U) and beta-D-galactosylqueuosine (Q*). The molecule also has a pseudouridine in the middle position of the anticodon, and is the first tRNA sequenced which has an adjacent pair of N2,N2-dimethylguanosine (m22G) residues.     

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.     

Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.     

The characterization of phosphoseryl tRNA from lactating bovine mammary gland.

BD-cellulose and RPC-5 chromatography of tRNA isolated from lactating bovine mammary gland showed the presence of four seryl-tRNA isoacceptors. The species, tRNA IV Ser, with the strongest affinity for BD-cellulose (required ethanol in the elution buffer) could be phosphorylated in the presence of serine, [gamma-32 P]-ATP, seryl-tRNA synthetase and phosphotransferase activity from the same tissue. O-Phosphoserine was identified as the 32P-labelled product after mild alkaline hydrolysis of this aminoacylated tRNA. Pancreatic ribonuclease treatment of the aminoacylated tRNA yielded a labelled product which was identified as phosphoseryladenosine. These results indicated there is a specific phosphoseryl tRNA species in lactating bovine mammary gland. It appears that the formation of phosphoseryl-tRNA proceeds by enzymic phosphorylation of seryl-tRNA.     

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster

The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.     

Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.