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
Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes ...Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs).Previously,two mutations(c.1129 G>A and c.517 T>C)were identified that led to hearing impairment;however,the underlying biochemical mechanism is unclear.In the present study,we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol,but affect the cytosolic LysRS level,its tertiary structure,and cytosolic tRNA aminoacylation in vitro.As for mitochondrial translation,the two mutations have little effect on the steady-state level,mitochondrial targeting,and tRNA binding affinity of mitochondrial LysRS.However,they exhibit striking differences in charging mitochondrial tRNALys,with the c.517T>C mutant being completely deficient in vitro and in vivo.We constructed two yeast genetic models,which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels.Overall,our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.展开更多
Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders.Recurrent mutations,including c.5A>G,p.D2G;c.1367C>T,p.S456L;c.1535G>A,p.R512Q an...Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders.Recurrent mutations,including c.5A>G,p.D2G;c.1367C>T,p.S456L;c.1535G>A,p.R512Q and c.1846_1847del,p.Y616Lfs*6 of RARS1 gene,which encodes two forms of human cytoplasmic arginyl-tRNA synthetase(hArgRS),are linked to Pelizaeus-Merzbacher-like disease(PMLD)with unclear pathogenesis.Among these mutations,c.5A>G is the most extensively reported mutation,leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS.Here,we showed the detrimental effects of R512Q substitution andΔC mutations on the structure and function of hArgRS,while the most frequent mutation c.5A>G,p.D2G acted in a different manner without impairing hArgRS activity.The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA,and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF.Taken together,our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.展开更多
Heme,as a prosthetic group of proteins,is an iron-protoporphyrin involved in a wide range of cellular functions.Cellular heme levels vary due to the accurate balance of its synthesis and degradation.The“heme sensor p...Heme,as a prosthetic group of proteins,is an iron-protoporphyrin involved in a wide range of cellular functions.Cellular heme levels vary due to the accurate balance of its synthesis and degradation.The“heme sensor protein”is currently a focus of investigation because heme has been found as a cellular signaling messenger involved in various biologic processes,including gene expression,protein localization,protein stability and microRNA processing.Several eukaryotic transcriptional factors can be regulated by heme,including heme activator protein(Hap1),Bach1,REV-erbα,and neuronal PAS domain protein 2(NPAS2).Especially,the two circadian transcrip-tional factors serving as the heme sensor,REV-erbαand NPAS2,coordinate the circadian clock with metabolic pathways.It is well established that heme regulates the activity of heme-regulated eukaryotic initiation factor 2α(eIF2α)kinase(HRI),which serves as a feedback inhibitor of protein translation in both erythroid and non-erythroid cells.Additionally,heme is involved in protein degradation by inducing the degradation of several proteins such as the iron response regulator(Irr),iron regulatory protein 2(IRP2),Bach1,and circadian factor period 2(Per2).The N-end rule ubiquitin-dependent protein degradation path-way has also been identified as a sensor of heme,which blocks the function of arginyl-tRNA protein transferase(ATE1)and E3 ubiquitin ligase.In this review,we summarize the regulatory roles of heme at the levels of transcription,protein translation,and protein degradation,highlighting the role of heme in maintaining cellular homeostasis.展开更多
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.
基金supported by the National Key Research and Development Program of China(2021YFA1300800 and 2021YFC2700903)the National Natural Science Foundation of China(32271300,91940302,31900436,and 81870896)+1 种基金the Committee of Science and Technology in Shanghai(22ZR1481300 and 22JC1400503)the Chinese Academy of Sciences(CAS)Project for Young Scientists in Basic Research(YSBR-075).
基金supported by the National Key Research and Development Program of China(2017YFA0504000)the National Natural Science Foundation of China(91940302,31500644,31570792,31822015,81870896,31670801)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19010203)Shanghai Key Laboratory of Embryo Original Diseases(Shelab201904)。
文摘Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs).Previously,two mutations(c.1129 G>A and c.517 T>C)were identified that led to hearing impairment;however,the underlying biochemical mechanism is unclear.In the present study,we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol,but affect the cytosolic LysRS level,its tertiary structure,and cytosolic tRNA aminoacylation in vitro.As for mitochondrial translation,the two mutations have little effect on the steady-state level,mitochondrial targeting,and tRNA binding affinity of mitochondrial LysRS.However,they exhibit striking differences in charging mitochondrial tRNALys,with the c.517T>C mutant being completely deficient in vitro and in vivo.We constructed two yeast genetic models,which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels.Overall,our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.
基金supported by the National Key Research and Development Program of China(2017YFA0504000)the Natural Science Foundation of China(91940302,31500644,31570792,31822015,81870896,31670801,31870811)+2 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19010203)Key Laboratory of Reproductive Genetics(Zhejiang University),Ministry of Education,P.R.China(ZDFY2020-RG-0003)Shanghai Key Laboratory of Embryo Original Diseases(Shelab201904).
文摘Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders.Recurrent mutations,including c.5A>G,p.D2G;c.1367C>T,p.S456L;c.1535G>A,p.R512Q and c.1846_1847del,p.Y616Lfs*6 of RARS1 gene,which encodes two forms of human cytoplasmic arginyl-tRNA synthetase(hArgRS),are linked to Pelizaeus-Merzbacher-like disease(PMLD)with unclear pathogenesis.Among these mutations,c.5A>G is the most extensively reported mutation,leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS.Here,we showed the detrimental effects of R512Q substitution andΔC mutations on the structure and function of hArgRS,while the most frequent mutation c.5A>G,p.D2G acted in a different manner without impairing hArgRS activity.The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA,and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF.Taken together,our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.
基金funded by the National Key Basic Research Foundation of China(No.2006CB910301)the National Natural Science Foundation of China(Grant No.30930022)+1 种基金the Postdoctoral Science Foundation of China,the Postdoctoral Foundation of Shanghai,the Postdoctoral Foundation of Shanghai Institutes for Biological Sciences(SIBS)K.C.Wong Postdoctoral Fellowship of the Chinese Academy of Sciences,and Sanofi-Aventis-SIBS Postdoctoral Fellowship.
文摘Heme,as a prosthetic group of proteins,is an iron-protoporphyrin involved in a wide range of cellular functions.Cellular heme levels vary due to the accurate balance of its synthesis and degradation.The“heme sensor protein”is currently a focus of investigation because heme has been found as a cellular signaling messenger involved in various biologic processes,including gene expression,protein localization,protein stability and microRNA processing.Several eukaryotic transcriptional factors can be regulated by heme,including heme activator protein(Hap1),Bach1,REV-erbα,and neuronal PAS domain protein 2(NPAS2).Especially,the two circadian transcrip-tional factors serving as the heme sensor,REV-erbαand NPAS2,coordinate the circadian clock with metabolic pathways.It is well established that heme regulates the activity of heme-regulated eukaryotic initiation factor 2α(eIF2α)kinase(HRI),which serves as a feedback inhibitor of protein translation in both erythroid and non-erythroid cells.Additionally,heme is involved in protein degradation by inducing the degradation of several proteins such as the iron response regulator(Irr),iron regulatory protein 2(IRP2),Bach1,and circadian factor period 2(Per2).The N-end rule ubiquitin-dependent protein degradation path-way has also been identified as a sensor of heme,which blocks the function of arginyl-tRNA protein transferase(ATE1)and E3 ubiquitin ligase.In this review,we summarize the regulatory roles of heme at the levels of transcription,protein translation,and protein degradation,highlighting the role of heme in maintaining cellular homeostasis.