线粒体RNAm^(3)C修饰酶METTL8的活力依赖于亚型特异的N-末端延伸结构且METTL8能修饰多种非天然底物tRNA

定位于线粒体的METTL8-Iso1和分布在核仁的METTL8-Iso4均为甲基转移酶基因METTL8通过mRNA选择性剪接产生的亚型,它们的区别仅为前者有N-末端延伸结构.METTL8-Iso1的功能是催化产生线粒体tRNATh和tRNASe(UCN)第32位3-甲基胞苷(mC32)修饰.METTL8-Iso4是否也具有tRNAmC32修饰活性以及N-末端延伸在线粒体tRNAmC32修饰中的作用尚不清楚。我们发现,N-末端延伸上有几个保守的关键氨基酸残基,而METTL8-Iso4由于缺少N-未端延伸而不具备tRNAmC32修饰活力.体外实验和体内实验的结果表明,甲基转移酶METTL2A和Trm140上对应的这些关键位点也是各自对底物tRNAmC32修饰活性所必需的。在跨细胞区室与跨物种的酶对tRNA体外催化的交又实验中,我们意外地发现METTL8-Iso1也能催化几种人细胞质tRNA甚至大肠杆菌tRNA的mC32修饰。此外,mC32修饰并不影响tRNA的N-苏氨酰氨基甲酰腺苷(t'A)修饰和氨基酰化活力.除了METTL8与人线粒体丝氨酰-tRNA合成酶(SARS2)的相互作用外,我们还进一步发现了人线粒体苏氨酰-tRNA合成酶(TARS2)与METTL8-IsoI之间的相互作用。体外实验中,METTL8-Iso1显著促进了SARS2和TARS2的氨酰化活力,表明线粒体IRNA的修饰和氨基酰化之间存在功能联系:总之,该结果加深了对线粒体mC32修饰生物发生机制的理解,并提供了一种制备仅含mC32修饰的人细胞质或细菌tRNA的方法,有助于未来研究mC32修饰对tRNA结构和功能的影响。

Human TRMT1 catalyzes m^(2)G or(m^(2))_(2)G formation on tRNAs in a substrate-dependent manner

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.

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNA^(Lys)

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.

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease

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.

Heme regulates protein homeostasis at transcription, protein translation, and degradation levels

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.