Mammalian ALKBH8 Possesses tRNA Methyltransferase Activity Required for the Biogenesis of Multiple Wobble Uridine Modifications Implicated in Translational Decoding

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm⁵U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8^−/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm⁵U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm⁵Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8^−/− mice appear normal. However, the selenocysteine-specific tRNA (tRNA^Sec) is aberrantly modified in the Alkbh8^−/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.tRNAs are frequently modified at the wobble uridine, a feature that is believed to either promote or restrict wobbling depending on the type of modification. In the case of eukaryotes, the functions of wobble uridine modifications have been studied in the greatest detail in Saccharomyces cerevisiae. Here, the modifications 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), and 5-carbamoylmethyluridine (ncm⁵U) or its 2′-O-ribose-methylated form, ncm⁵Um, are found in 11 out of 13 wobble uridine-containing tRNAs (22). mcm⁵U and mcm⁵s²U are mostly found in “split” codon boxes, where the pyrimidine- and purine-ending codons encode different amino acids, while ncm⁵U is found in “family” codon boxes, where all four codons encode a single amino acid. Early reports based on in vitro experiments suggested that wobble nucleosides, such as mcm⁵U, ncm⁵U, and their derivatives, may restrict wobbling (17, 37, 45), but the results of a recent comprehensive study performed in vivo in S. cerevisiae show that such modifications can improve the reading both of the cognate, A-ending codons and of the wobble, G-ending codons (22). This may suggest that the primary role of these modified nucleosides is to improve translational efficiency rather than to restrict wobbling.The characterization of wobble uridine modifications in higher eukaryotes is very limited, and little is known about the enzymes that introduce them. In mammals, mcm⁵s²U has been found in the wobble position of tRNA^Glu(UUC), tRNA^Lys(UUU), and tRNA^Arg(UCU) (40). Unlike yeast, mammals possess a specialized tRNA that is responsible for recoding the UGA stop codon to insert the 21st amino acid, selenocysteine (Sec). The mammalian tRNA^Sec population consists of two subpopulations containing either mcm⁵U or the ribose-methylated derivative mcm⁵Um in the wobble position. Interestingly, ribose methylation of mcm⁵U in tRNA^Sec appears to have a role in regulating selenoprotein synthesis, as the expression of some selenoproteins, such as glutathione peroxidase 1 (Gpx1), appears to be promoted by mcm⁵Um-containing tRNA^Sec (5, 7, 9, 32).Some years ago, the Escherichia coli AlkB protein was found to be a 2-oxoglutarate- and iron-dependent dioxygenase capable of demethylating the lesions 1-methyladenosine and 3-methylcytosine in DNA (13, 42). Multicellular organisms generally possess several different AlkB homologues (ALKBH), and bioinformatics analysis has identified eight different mammalian ALKBH proteins, denoted ALKBH1 to ALKBH8 in humans and Alkbh1 to Alkbh8 in mice, as well as the somewhat-less-related, obesity-associated FTO protein (2, 16, 30). Among the ALKBH proteins of unknown function, ALKBH8 is the only one containing additional annotated protein domains. Here, the AlkB domain is localized between an N-terminal RNA recognition motif (RRM) and a C-terminal methyltransferase (MT) domain. Interestingly, the MT domain has sequence homology to the S. cerevisiae tRNA methyltransferase Trm9, which has been shown to catalyze the methyl esterification of modified wobble uridine (U34) residues of tRNA^Arg and tRNA^Glu, resulting in the formation of mcm⁵U and mcm⁵s²U, respectively (23, 43). Until recently, human ALKBH8 was incorrectly annotated in the protein sequence database, and another human protein, KIAA1456, has been designated the human Trm9 homologue (3, 23).We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm⁵U, mcm⁵s²U, and mcm⁵Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm⁵U) and/or the amide forms ncm⁵U/ncm⁵s²U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm⁵U and cm⁵s²U to mcm⁵U and mcm⁵s²U, respectively. In agreement with the involvement of mcm⁵Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8^−/− mice, and tRNA^Sec from these mice showed a reduced ability to decode the UGA stop codon to Sec.     

玉米黑粉菌DNA载体的构建和它的原生质球的转化和再生

我们用玉米黑粉菌(Ustilago maydis)热休克基因(Heat shock gene,hsp 70)的启动子和终止子与新霉素磷酸转移酶基因相连,构建成了有效的双功能质粒pDL1(在E。coli中用Amp作为选择标记,在玉米黑粉菌中用新霉素作为选择标记)。以分离的一株新霉素敏感的玉米黑粉菌作为受体菌,用构建的pDL1质粒作为供体DNA,对影响受体菌原生质球的形成条件(培养基、菌龄、各种酶、酶的浓度、作用时间和渗透压稳定剂)、再生条件和DNA转化条件进行了初步研究。对数前中期收集的菌体,以5mg/ml真菌溶壁酶处理30分钟左右,90%都形成原生质球,其再生率为60—80%,转化率为300—1000转化子/μg DNA。随机选出25个转化子,DNA杂交都为阳性。分析dot-blot和Southern blot DNA杂交结果,发现质粒在细胞中以整合形式存在,一个细胞可以有多拷贝的整合质粒,质粒可能以非完全同源性的重组形式,参入性地整合进染色体中。     

Externally added aFGF mutants do not require extensive unfolding for transport to the cytosol and the nucleus in NIH/3T3 cells

Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.     

Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment

A number of proteins are able to enter cells from the extracellular environment, including protein toxins, growth factors, viral proteins, homeoproteins, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence from the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment.The fusion of the TAT-peptide to dtA converted the protein to a heparin-binding protein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficiently deliver enzymatically active dtA to the cytosol. Interestingly, the fused TAT-peptide potentiated the binding and cytotoxic effect of the corresponding holotoxin. We made a fusion protein between VP22, another membrane-permeant protein, and dtA, and also in this case we detected association with cells in the absence of a cytotoxic effect. The data indicate that transport of dtA into the cell by the TAT-peptide and VP22 is inefficient.     

Protein methylation in mitochondria

Many proteins are modified by posttranslational methylation, introduced by a number of methyltransferases (MTases). Protein methylation plays important roles in modulating protein function and thus in optimizing and regulating cellular and physiological processes. Research has mainly focused on nuclear and cytosolic protein methylation, but it has been known for many years that also mitochondrial proteins are methylated. During the last decade, significant progress has been made on identifying the MTases responsible for mitochondrial protein methylation and addressing its functional significance. In particular, several novel human MTases have been uncovered that methylate lysine, arginine, histidine, and glutamine residues in various mitochondrial substrates. Several of these substrates are key components of the bioenergetics machinery, e.g., respiratory Complex I, citrate synthase, and the ATP synthase. In the present review, we report the status of the field of mitochondrial protein methylation, with a particular emphasis on recently discovered human MTases. We also discuss evolutionary aspects and functional significance of mitochondrial protein methylation and present an outlook for this emergent research field.     

Success of maximum likelihood phylogeny inference in the four-taxon case

We used simulated data to investigate a number of properties of maximum- likelihood (ML) phylogenetic tree estimation for the case of four taxa. Simulated data were generated under a broad range of conditions, including wide variation in branch lengths, differences in the ratio of transition and transversion substitutions, and the absence of presence of gamma-distributed site-to-site rate variation. Data were analyzed in the ML framework with two different substitution models, and we compared the ability of the two models to reconstruct the correct topology. Although both models were inconsistent for some branch-length combinations in the presence of site-to-site variation, the models were efficient predictors of topology under most simulation conditions. We also examined the performance of the likelihood ratio (LR) test for significant positive interior branch length. This test was found to be misleading under many simulation conditions, rejecting too often under some simulation conditions. Under the null hypothesis of zero length internal branch, LR statistics are assumed to be asymptotically distributed chi 2(1); with limited data, the distribution of LR statistics under the null hypothesis varies from chi 2(1).      

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02–0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.