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1.
Here, we have investigated the specificity of purified recombinant tRNA:m(2)(2)G10 methyltransferase of Pyrococcus abyssi ((Pab)Trm-m(2)(2)G10 enzyme). This archaeal enzyme catalyses mono- and dimethylation of the N(2)-exocyclic amino group of guanine at position 10 of several tRNA species. Our results indicate that only few identity elements are required for the efficient formation of m(2)(2)G10. They are composed of a G10.U25 wobble base-pair in the dihydrouridine arm (D-arm) and a four nucleotide variable loop (V-loop) within a canonical three-dimensional (3D) structure. The types of base-pairs in the D-arm or amino acid acceptor stem are also important for the enzymatic reaction, but appear to affect only the rate of tRNA methylation. However, in tRNA species harbouring a G10-C25 Watson-Crick base-pair and/or five nucleotide V-loop, only m(2)G10 is produced. To impair the monomethylation reaction, drastic amputation in the T-arm is required. Our observations contrast with those reported earlier for the identity elements required for a remotely related Pyrococcus furiosus Trm-m(2)(2)G26 enzyme (alias (Pfu)Trm1) that also catalyses the two step formation of m(2)(2)G but at position 26 in several tRNA species. In this case, a G10-C25 base-pair together with the five nucleotide V-loop were shown to be required for efficient formation of m(2)(2)G26. Thus, in the Pyrococcus genus, the major identity elements that preclude formation of m(2)(2)G at positions 10 or 26 in tRNA are mutually exclusive. Therefore, the Trm-m(2)(2)G10 and Trm-m(2)(2)G26 enzymes have evolved independently towards different specificities. In addition, identity elements for m(2)/m(2)(2)G10 formation in archaeal tRNA are different from the ones required for m(2)G10 formation in eukaryal tRNA. We propose that archaeal tRNA:m(2)(2)G10 methyltransferases, unlike the orthologous eukaryal tRNA:m(2)G10 methyltransferases, evolved towards m(2)(2)G10 specificity due to the possible requirement of preventing formation of alternative structures in G/C rich archaeal tRNA species.  相似文献   

2.
The N1-methyl-Adenosine (m1A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m1A58 modification is essential for viability because it is required for the stability of the initiator-tRNAMet. However, m1A58 modification is not required for the stability of several other tRNAs in yeast. This differential m1A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m1A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m1A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNAMet is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m1A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m1A58 hypomodified tRNAs. Most m1A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m1A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.  相似文献   

3.
In human mitochondria, 1-methyladenosine (m1A) occurs at position 58 of tRNALeu(UUR). In addition, partial m1A58 modifications have been found in human mitochondrial tRNALys and tRNASer(UCN). We identified human Trmt61B, which encodes a mitochondria-specific tRNA methyltransferase responsible for m1A58 in these three tRNAs. Trmt61B is dominantly localized to the mitochondria. m1A58 formation in human mitochondrial tRNALeu(UUR) could be reconstituted in vitro using recombinant Trmt61B in the presence of Ado-Met as a methyl donor. Unlike the cytoplasmic tRNA m1A58 methyltransferase that consists of an α2β2 heterotetramer formed by Trmt61A and Trmt6, Trmt61B formed a homo-oligomer (presumably a homotetramer) that resembled the bacterial homotetrameric m1A58 methyltransferase. The bacterial origin of Trmt61B is supported by the results of the phylogenetic analysis.  相似文献   

4.
5.
Ero R  Peil L  Liiv A  Remme J 《RNA (New York, N.Y.)》2008,14(10):2223-2233
In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m3Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m3Ψ1915 is the only methylated pseudouridine in bacteria described to date.  相似文献   

6.
Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.  相似文献   

7.
Pseudouridine synthase 1 (Pus1p) is an enzyme that converts uridine to Pseudouridine (Ψ) in tRNA and other RNAs in eukaryotes. The active site of Pus1p is composed of stretches of amino acids that are highly conserved and it is hypothesized that mutation of select residues would impair the enzyme's ability to catalyze the formation of Ψ. However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all Ψ synthases tested. To determine the requirements for particular amino acids at certain absolutely conserved positions in Pus1p, three residues (R116, Y173, R267) that correspond to amino acids known to compose the active site of TruA, a bacterial Ψ synthase that is homologous to Pus1p, were mutated in human Pus1p (hPus1p). The effects of those mutations were determined with three different in vitro assays of pseudouridylation and several tRNA substrates. Surprisingly, it was found that each of these components of the hPus1p active site could tolerate certain amino acid substitutions and in fact most mutants exhibited some activity. The most active mutants retained near wild-type activity at positions 27 or 28 in the substrate tRNA, but activity was greatly reduced or absent at other positions in tRNA readily modified by wild-type hPus1p.  相似文献   

8.
Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m5C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m5C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNACys and tRNAThr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.  相似文献   

9.
Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.  相似文献   

10.
Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2′-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams–Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3′-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3′-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N7-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.  相似文献   

11.
12.
The presence of tricyclic wyosine derivatives 3′-adjacent to anticodon is a hallmark of tRNAPhe in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N1-methylation of guanosine and C7-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m1G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNAPhe, is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.  相似文献   

13.
N1-Acetylnorspermidine [CH3CONH(CH2)3 NH(CH2)3NH3] was identified in Vibrio parahaemolyticus, which contains norspermidine as a major polyamine. This is the first example for the natural occurrence of monoacetylated unusual polyamine. The N1-acetylnorspermidine content was the highest 4 h after inoculation. Incubation of norspermidine and acetyl CoA with a cell extract from V. parahaemolyticus produced N1-acetylnorspermidine. A remarkable increase in specific activity of the acetyltransferase was observed at the exponential phase of growth. Spermidine also served as a substrate for the enzyme, with the formation of two isomers of the acetylspermidines (N1-acetylspermidine was predominant), but the reaction rate was less than 50% of that with norspermidine. These results suggest that norspermidine in V. parahaemolyticus may be associated with the cell growth and its role may be controlled through acetylation, as reported for spermidine in Escherichia coli.  相似文献   

14.
Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo5UAC) contains a unique modification, N6-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA1Val. Inactivation of yfiC gene abolishes m6A formation in tRNA1Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA1Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m6A in tRNA1Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.  相似文献   

15.
The ribonuclease P (RNase P) proteins TkoPop5 and TkoRpp30, homologs of human Pop5 and Rpp30, respectively, in the hyperthermophilic archaeon Thermococcus kodakarensis were prepared and characterized with respect to pre-tRNA cleavage activity using the reconstitution system of the well-studied Pyrococcus horikoshii RNase P. The reconstituted particle containing TkoPop5 in place of the P. horikoshii counterpart PhoPop5 retained pre-tRNA cleavage activity comparable to that of the reconstituted P. horikoshii RNase P, while that containing TkoRpp30 instead of its corresponding protein PhoRpp30 had slightly lower activity than the P. horikoshii RNase P. Moreover, we determined crystal structures of TkoRpp30 alone and in complex with TkoPop5. Like their P. horikoshii counterparts, whose structures were solved previously, TkoRpp30 and TkoPop5 fold into TIM barrel and RRM-like fold, respectively. This finding demonstrates that RNase P proteins in T. kodakarensis and P. horikoshii are interchangeable and that their three-dimensional structures are highly conserved.  相似文献   

16.
Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm5s2U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm5s2U and Ψ38 in tRNAGlnUUG was shown to mediate efficient synthesis of the Q/N rich [PIN+] prion forming protein Rnq1.1 Klassen R, Ciftci A, Johanna Funk J, Bruch A, Butter F, Schaffrath R. tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast. Nucleic Acids Res 2016; 44(22):10946-959. pii: gkw705; PMID:27496282; http://dx.doi.org/10.1093/nar/gkw705[Crossref], [PubMed], [Web of Science ®] [Google Scholar] In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNAGlnUUG, but not its isoacceptor tRNAGlnCUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm5s2U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.  相似文献   

17.
Kay Hofmann 《EMBO reports》2008,9(12):1196-1202
The ubiquitin‐like protein Urm1 can be covalently conjugated to other proteins, such as the yeast thioredoxin peroxidase protein Ahp1p, through a mechanism involving the ubiquitin E1‐like enzyme Uba4. Recent findings have revealed a second function of Urm1 as a sulphur carrier in the thiolation of eukaryotic cytoplasmic transfer RNAs (tRNAs). Interestingly, this new role of Urm1 is similar to the sulphur‐carrier activity of its prokaryotic counterparts, strengthening the hypothesis that Urm1 is a molecular fossil of the ubiquitin‐like protein family. Here, we discuss the function of Urm1 in light of its dual role in protein and RNA modification.  相似文献   

18.
19.
虎源H5N1亚型禽流感病毒感染小鼠模型的建立   总被引:6,自引:0,他引:6  
为研究H5N1亚型禽流感病毒的病原特性、致病机理及对其疫苗与救治药物效果评价提供平台,利用本室分离鉴定的虎源A/Tiger/Harbin/01/2002株(简称HAB/01)H5N1亚型禽流感病毒进行连续10倍稀释后,对4~6周龄 雄性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定其MLD50,检测小鼠感染、致病的多项指标,观察期为14d.结果感染小鼠呈现出规律的以肺炎为主的临床症状、病理变化及病死率;测得该病毒对小鼠的MLD50为10-7.1/0.05mL.成功建立了虎源H5N1亚型禽流感病毒感染BALB/c小鼠的实验模型.  相似文献   

20.
2004年1月湖北宜昌某鸡场暴发疫病,从该鸡场濒死鸡肺组织中分离到了一株病毒,电镜切片观察到典型的禽流感病毒粒子;采用ELISA检测禽流感抗原为阳性;RT-PCR扩增HA、NA基因并测序,经BLAST分析,HA基因与A/Goose/Guangdong/1/96(H5N1)HA基因同源性为97%;NA基因与A/Goose/Guangdong/1/96(H5N1)NA基因同源性为96%,确定该分离株为禽流感病毒H5N1亚型(A/Chicken/Yichang/Lung-1/04(H5N1))。  相似文献   

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