Selenocysteine (Sec) tRNAs serve as carrier molecules for the biosynthesis of Sec from serine and to donate Sec to protein in response to specific UGA codons. In this study, we describe the current status of Sec tRNAs...Selenocysteine (Sec) tRNAs serve as carrier molecules for the biosynthesis of Sec from serine and to donate Sec to protein in response to specific UGA codons. In this study, we describe the current status of Sec tRNAs in higher animals and further we exarnine: (i) the Sec tRNA population in Drosophila; (ii) transcription of the Sec tRNA in vivo (in Xenopus oocytes) and in vitro (in Xenopus oocyte extracts); (iii) the effect of selenium on the Sec tRNA population in various rat tissues following replenishment of extremely selenium deficient rats with this element; and (iv) the biosynthesis of the modified bases on Sec tRNA in Xenopus oocytes展开更多
Codon nonsense mutations include amber, ochre, or opal mutations according to termination codon consisting of three types (TAG, TAA and TGA). Codon nonsense mutations are also divided into natural and artificial mutat...Codon nonsense mutations include amber, ochre, or opal mutations according to termination codon consisting of three types (TAG, TAA and TGA). Codon nonsense mutations are also divided into natural and artificial mutations. We discussed the interaction of codon nonsense mutations and suppressor tRNAs in vitro and in vivo. Nonsense suppressions do not only happen in prokaryotes but also in eukaryotes. Meanwhile, the misreading of termination codon and in-corporation of nonnatural amino acids into proteins are also introduced.展开更多
Using purified bovine liver tRNA￣(Ile) and yeast tRNA￣(Phe), we studied the effects of spermine on the melting curve and melting temperature(Tm) of the tRNAs. The results showed that the absorbance at 260 nm of tRNA...Using purified bovine liver tRNA￣(Ile) and yeast tRNA￣(Phe), we studied the effects of spermine on the melting curve and melting temperature(Tm) of the tRNAs. The results showed that the absorbance at 260 nm of tRNAs decreases with the increase of temperature in the presence of 2 mmol/L spermine. We called this phenomenon hypochromism and reverse-Tm of the tRNAs. It is suggested that spermine binds to tRNAs and stabilizes the secondary structure of the tRNAs.展开更多
TRMT1 is an N^2-methylguanosine(m^(2)G)and N^2,N^2-methylguanosine((m^(2))_(2)G)methyltransferase that targets G26 of both cytoplasmic and mitochondrial t RNAs.In higher eukaryotes,most cytoplasmic t RNAs with G26 car...TRMT1 is an N^2-methylguanosine(m^(2)G)and N^2,N^2-methylguanosine((m^(2))_(2)G)methyltransferase that targets G26 of both cytoplasmic and mitochondrial t RNAs.In higher eukaryotes,most cytoplasmic t RNAs with G26 carry(m^(2))_(2)G26,although the majority of mitochondrial G26-containing t RNAs carry m^(2)G26 or G26,suggesting differences in the mechanisms by which TRMT1 catalyzes modification of these t RNAs.Loss-of-function mutations of human TRMT1 result in neurological disorders and completely abrogate t RNA:(m^(2))_(2)G26 formation.However,the mechanism underlying the independent catalytic activity of human TRMT1 and identity of its specific substrate remain elusive,hindering a comprehensive understanding of the pathogenesis of neurological disorders caused by TRMT1 mutations.Here,we showed that human TRMT1 independently catalyzes formation of the t RNA:m^(2)G26 or(m^(2))_(2)G26 modification in a substrate-dependent manner,which explains the distinct distribution of m^(2)G26 and(m^(2))_(2)G26 on cytoplasmic and mitochondrial t RNAs.For human TRMT1-mediated t RNA:(m^(2))_(2)G26 formation,the semi-conserved C11:G24 serves as the determinant,and the U10:A25 or G10:C25 base pair is also required,while the size of the variable loop has no effect.We defined the requirements of this recognition mechanism as the“(m^(2))_(2)G26 criteria”.We found that the(m^(2))_(2)G26 modification occurred in almost all the higher eukaryotic t RNAs conforming to these criteria,suggesting the“(m^(2))_(2)G26 criteria”are applicable to other higher eukaryotic t RNAs.展开更多
为了解拟南芥中Dicer-like蛋白对tRNA衍生的小RNA(tRNA-derived small RNAs,tsRNAs)的产生有何影响,对拟南芥野生型和不同Dicer-like(DCL)基因突变体进行tRNA-seq测序,并分析tsRNA和tRNA的表达量.结果显示,DCL4基因突变后tsRNA的表达量...为了解拟南芥中Dicer-like蛋白对tRNA衍生的小RNA(tRNA-derived small RNAs,tsRNAs)的产生有何影响,对拟南芥野生型和不同Dicer-like(DCL)基因突变体进行tRNA-seq测序,并分析tsRNA和tRNA的表达量.结果显示,DCL4基因突变后tsRNA的表达量明显降低,说明DCL4可能参与tsRNA的产生.拟南芥tRC1位点(Chr1:21268000-21310000)具有大量串联分布的tRNA序列,通过对RNA介导的甲基化(RdDM)途径相关基因CLSY1突变体中tRC1位点的24 nt siRNA和tsRNA进行分析,推断tRC1位点的tsRNA受RdDM途径负调控.综上,本研究鉴定到DCL4在tsRNA生成中的潜在作用,部分tsRNA的生成与RdDM途径有关.展开更多
Transfer RNA-derived small RNAs(tsRNAs)have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells.However,it is unknown whether tsRNAs also regulate...Transfer RNA-derived small RNAs(tsRNAs)have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells.However,it is unknown whether tsRNAs also regulate embryo hatching.In this study,we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA(miRNA)cargo of preimplantation embryonic extracellular vesicles(EVs)influences embryo development.We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts.The majority of tsRNAs was identified as tRNA halves originating from the 5'ends of tRNAs.Among the 148 differentially expressed tsRNAs,the 19 nt tRNA fragment(tRF)tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts.RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group(P<0.05).Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching(P<0.05).Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation.In summary,tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions,and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching,while influencing embryo implantation-related genes and pathways.These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.展开更多
BACKGROUND Mutations in mitochondrial tRNA(mt-tRNA)genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus(T2DM).We previously reported a large Chinese pedigree with maternally ...BACKGROUND Mutations in mitochondrial tRNA(mt-tRNA)genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus(T2DM).We previously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel mt-tRNA^(Trp)A5514G and tRNA^(Ser(AGY))C12237T variants,however,the effects of these mt-tRNA variants on T2DM progression are largely unknown.AIM To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic,molecular,and biochemical levels.METHODS Cytoplasmic hybrid(cybrid)cells carrying both m.A5514G and m.C12237T variants,and healthy control cells without these mitochondrial DNA(mtDNA)variants were generated using trans-mitochondrial technology.Mitochondrial features,including mt-tRNA steady-state level,levels of adenosine triphosphate(ATP),mitochondrial membrane potential(MMP),reactive oxygen species(ROS),mtDNA copy number,nicotinamide adenine dinucleotide(NAD+)/NADH ratio,enzymatic activities of respiratory chain complexes(RCCs),8-hydroxy-deoxyguanine(8-OhdG),malondialdehyde(MDA),and superoxide dismutase(SOD)were examined in cell lines with and without these mt-tRNA variants.RESULTS Compared with control cells,the m.A5514G variant caused an approximately 35%reduction in the steady-state level of mt-tRNA^(Trp)(P<0.0001);however,the m.C12237T variant did not affect the mt-tRNA^(Ser(AGY))steady-state level(P=0.5849).Biochemical analysis revealed that cells with both m.A5514G and m.C12237T variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells:ATP,MMP,NAD+/NADH ratio,enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells(P<0.05 for all measures).By contrast,the levels of ROS,8-OhdG and MDA were significantly increased(P<0.05 for all measures),but mtDNA copy number was not affected by m.A5514G and m.C12237T variants(P=0.5942).CONCLUSION The m.A5514G variant impaired mt-tRNA^(Trp)metabolism,which subsequently caused mitochondrial dysfunction.The m.C12237T variant did not alter the steady-state level of mt-tRNA^(Ser(AGY)),indicating that it may be a modifier of the m.A5514G variant.The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.展开更多
文摘Selenocysteine (Sec) tRNAs serve as carrier molecules for the biosynthesis of Sec from serine and to donate Sec to protein in response to specific UGA codons. In this study, we describe the current status of Sec tRNAs in higher animals and further we exarnine: (i) the Sec tRNA population in Drosophila; (ii) transcription of the Sec tRNA in vivo (in Xenopus oocytes) and in vitro (in Xenopus oocyte extracts); (iii) the effect of selenium on the Sec tRNA population in various rat tissues following replenishment of extremely selenium deficient rats with this element; and (iv) the biosynthesis of the modified bases on Sec tRNA in Xenopus oocytes
文摘Codon nonsense mutations include amber, ochre, or opal mutations according to termination codon consisting of three types (TAG, TAA and TGA). Codon nonsense mutations are also divided into natural and artificial mutations. We discussed the interaction of codon nonsense mutations and suppressor tRNAs in vitro and in vivo. Nonsense suppressions do not only happen in prokaryotes but also in eukaryotes. Meanwhile, the misreading of termination codon and in-corporation of nonnatural amino acids into proteins are also introduced.
文摘Using purified bovine liver tRNA￣(Ile) and yeast tRNA￣(Phe), we studied the effects of spermine on the melting curve and melting temperature(Tm) of the tRNAs. The results showed that the absorbance at 260 nm of tRNAs decreases with the increase of temperature in the presence of 2 mmol/L spermine. We called this phenomenon hypochromism and reverse-Tm of the tRNAs. It is suggested that spermine binds to tRNAs and stabilizes the secondary structure of the tRNAs.
基金the National Key Research and Development Program of China(2021YFA1100800,2020YFA0803401)the National Natural Science Foundation of China(32022040,91940302,31971230,31870811,32000919)Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at Shanghai Tech University。
文摘TRMT1 is an N^2-methylguanosine(m^(2)G)and N^2,N^2-methylguanosine((m^(2))_(2)G)methyltransferase that targets G26 of both cytoplasmic and mitochondrial t RNAs.In higher eukaryotes,most cytoplasmic t RNAs with G26 carry(m^(2))_(2)G26,although the majority of mitochondrial G26-containing t RNAs carry m^(2)G26 or G26,suggesting differences in the mechanisms by which TRMT1 catalyzes modification of these t RNAs.Loss-of-function mutations of human TRMT1 result in neurological disorders and completely abrogate t RNA:(m^(2))_(2)G26 formation.However,the mechanism underlying the independent catalytic activity of human TRMT1 and identity of its specific substrate remain elusive,hindering a comprehensive understanding of the pathogenesis of neurological disorders caused by TRMT1 mutations.Here,we showed that human TRMT1 independently catalyzes formation of the t RNA:m^(2)G26 or(m^(2))_(2)G26 modification in a substrate-dependent manner,which explains the distinct distribution of m^(2)G26 and(m^(2))_(2)G26 on cytoplasmic and mitochondrial t RNAs.For human TRMT1-mediated t RNA:(m^(2))_(2)G26 formation,the semi-conserved C11:G24 serves as the determinant,and the U10:A25 or G10:C25 base pair is also required,while the size of the variable loop has no effect.We defined the requirements of this recognition mechanism as the“(m^(2))_(2)G26 criteria”.We found that the(m^(2))_(2)G26 modification occurred in almost all the higher eukaryotic t RNAs conforming to these criteria,suggesting the“(m^(2))_(2)G26 criteria”are applicable to other higher eukaryotic t RNAs.
文摘为了解拟南芥中Dicer-like蛋白对tRNA衍生的小RNA(tRNA-derived small RNAs,tsRNAs)的产生有何影响,对拟南芥野生型和不同Dicer-like(DCL)基因突变体进行tRNA-seq测序,并分析tsRNA和tRNA的表达量.结果显示,DCL4基因突变后tsRNA的表达量明显降低,说明DCL4可能参与tsRNA的产生.拟南芥tRC1位点(Chr1:21268000-21310000)具有大量串联分布的tRNA序列,通过对RNA介导的甲基化(RdDM)途径相关基因CLSY1突变体中tRC1位点的24 nt siRNA和tsRNA进行分析,推断tRC1位点的tsRNA受RdDM途径负调控.综上,本研究鉴定到DCL4在tsRNA生成中的潜在作用,部分tsRNA的生成与RdDM途径有关.
基金supported by Ghent University(Grant:Bijzonder Onderzoeksfonds Geconcerteerde Onderzoeksactie 2018000504[GOA030-18 BOF])supported by Ghent University:BOF.STG.2022.02.0034.01+1 种基金supported by China Scholarship Council:Grant 202006910034supported by Fonds Wetenschappelijk Onderzoek:Grant 1228821N and 12A2H24N。
文摘Transfer RNA-derived small RNAs(tsRNAs)have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells.However,it is unknown whether tsRNAs also regulate embryo hatching.In this study,we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA(miRNA)cargo of preimplantation embryonic extracellular vesicles(EVs)influences embryo development.We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts.The majority of tsRNAs was identified as tRNA halves originating from the 5'ends of tRNAs.Among the 148 differentially expressed tsRNAs,the 19 nt tRNA fragment(tRF)tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts.RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group(P<0.05).Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching(P<0.05).Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation.In summary,tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions,and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching,while influencing embryo implantation-related genes and pathways.These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.
基金Supported by the Hangzhou Joint Fund of the Zhejiang Provincial Natural Science Foundation of China,No.LHZY24H020002Hangzhou Municipal Health Commission,No.ZD20220010Quzhou Bureau of Science and Technology,No.2022K51.
文摘BACKGROUND Mutations in mitochondrial tRNA(mt-tRNA)genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus(T2DM).We previously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel mt-tRNA^(Trp)A5514G and tRNA^(Ser(AGY))C12237T variants,however,the effects of these mt-tRNA variants on T2DM progression are largely unknown.AIM To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic,molecular,and biochemical levels.METHODS Cytoplasmic hybrid(cybrid)cells carrying both m.A5514G and m.C12237T variants,and healthy control cells without these mitochondrial DNA(mtDNA)variants were generated using trans-mitochondrial technology.Mitochondrial features,including mt-tRNA steady-state level,levels of adenosine triphosphate(ATP),mitochondrial membrane potential(MMP),reactive oxygen species(ROS),mtDNA copy number,nicotinamide adenine dinucleotide(NAD+)/NADH ratio,enzymatic activities of respiratory chain complexes(RCCs),8-hydroxy-deoxyguanine(8-OhdG),malondialdehyde(MDA),and superoxide dismutase(SOD)were examined in cell lines with and without these mt-tRNA variants.RESULTS Compared with control cells,the m.A5514G variant caused an approximately 35%reduction in the steady-state level of mt-tRNA^(Trp)(P<0.0001);however,the m.C12237T variant did not affect the mt-tRNA^(Ser(AGY))steady-state level(P=0.5849).Biochemical analysis revealed that cells with both m.A5514G and m.C12237T variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells:ATP,MMP,NAD+/NADH ratio,enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells(P<0.05 for all measures).By contrast,the levels of ROS,8-OhdG and MDA were significantly increased(P<0.05 for all measures),but mtDNA copy number was not affected by m.A5514G and m.C12237T variants(P=0.5942).CONCLUSION The m.A5514G variant impaired mt-tRNA^(Trp)metabolism,which subsequently caused mitochondrial dysfunction.The m.C12237T variant did not alter the steady-state level of mt-tRNA^(Ser(AGY)),indicating that it may be a modifier of the m.A5514G variant.The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.
