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Extensive profiling of the expressions of trnas and trna-derived fragments(tRFs)reveals the complexities of trna and tRF populations in plants

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Science China(Life Sciences) 2021年第4期64卷 495-511页

作者： Xuan Ma Chunyan Liu Xiaocong Kong Jing Liu Siju Zhang Shanshan Liang Weijiang Luan Xiaofeng Cao Tianjin Key Laboratory of Animal and Plant Resistance College of Life SciencesTianjin Normal UniversinTianjin 300387China State Key Laboratory of Plant Genomics and National Center for Plant Gene Research Institute of Genetics and Developmental BiologyChinese Academy of Sciences.Beijing 100101China

Evidence is emerging that t RNA-derived fragments(t RFs)are regulatory molecules.Studies of t RFs in plants have been based on conventional small RNA sequencing,and focused on profiling of t RF-5 and t RF-3 species.A more comprehensive and quantitative analysis of the entire t RF population is highly necessary.Here,we employ t RNA-seq and YAMAT-seq,and develop a bioinformatics tool to comprehensively profile the expressions of t RNAs and t RFs in plants.We show that in Arabidopsis,approximately half of t RNA genes are extremely weakly expressed,accounting for only 1%of total t RNA abundance,while~12%of t RNA genes contribute to~80%of t RNA abundance.Our t RNA sequencings in various plants reveal that t RNA expression profiles exhibit a cross-species conserved pattern.By characterizing the composition of a highly heterogeneous t RF population,we show that t RNA halves and previously unnoticed 10–16-nt tiny t RFs represent substantial portions.The highly accumulated 13-nt and 16-nt tiny t RFs in Arabidopsis indicate that tiny t RFs are not random t RNA degradation products.Finally,we provide a user-friendly database for displaying the dynamic spatiotemporal expressions of t RNAs and t RFs in the model plants Arabidopsis and rice.

关键词： trna tRFs trna halves tiny tRFs trna-seq

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

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Science China(Life Sciences) 2020年第8期63卷 1227-1239页

作者： Yong Wang Jing-Bo Zhou Qi-Yu Zeng Siqi Wu Mei-Qin Xue Pengfei Fang En-Duo Wang Xiao-Long Zhou State Key Laboratory of Molecular Biology CAS Center for Excellence in Molecular Cell ScienceShanghai Institute of Biochemistry and Cell BiologyChinese Academy of SciencesUniversity of Chinese Academy of SciencesShanghai 200031China School of Life Science and Technology ShanghaiTech UniversityShanghai 201210China State Key Laboratory of Bioorganic and Natural Products Chemistry Center for Excellence in Molecular SynthesisShanghai Institute of Organic ChemistryChinese Academy of SciencesShanghai 200032China

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.

关键词： aminoacyl-trna synthetase trna aminoacylation protein synthesis

trna-sgRNA/Cas9系统介导多年生黑麦草原生质体的基因编辑

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草业学报 2023年第4期32卷 129-141页

作者：姚佳明郝欢欢张敬徐彬南京农业大学草业学院江苏南京210095

trna可以将多个sgRNAs连接起来合并成一个多顺反子基因,再与CRISPR/Cas9表达载体结合形成多顺反子trna-sgRNA/Cas9(PTG/Cas9)系统来对多靶点进行基因编辑。该系统已经在水稻中验证其可促进sgRNAs的转录和提高多靶点的编辑效率。因此,为... 详细信息

trna可以将多个sgRNAs连接起来合并成一个多顺反子基因,再与CRISPR/Cas9表达载体结合形成多顺反子trna-sgRNA/Cas9(PTG/Cas9)系统来对多靶点进行基因编辑。该系统已经在水稻中验证其可促进sgRNAs的转录和提高多靶点的编辑效率。因此,为了在多年生黑麦草中实现高效的基因编辑,本研究中,构建了2个带有trna的CRISPR中间载体,并提供了一种快速、灵活的PTG/Cas9载体构建方法。为了快速验证PTG/Cas9系统是否可以在多年生黑麦草基因组中发挥功能,用聚乙二醇4000(PEG 4000)介导PTG/Cas9质粒转化多年生黑麦草原生质体,后提取原生质体DNA扩增目的序列检测是否有目标基因被编辑的细胞。试验结果显示,PTG/Cas9系统可用于多年生黑麦草基因编辑,且基因编辑效率约为6.7%。试验结果为多年生黑麦草遗传研究和育种提供了重要的基础。

关键词： CRISPR/Cas9 trna 多基因编辑载体质粒原生质体多年生黑麦草

The occurrence order and cross-talk of different trna modifications

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Science China(Life Sciences) 2021年第9期64卷 1423-1436页

作者： Jing Li Wen-Yu Zhu Wen-Qing Yang Cai-Tao Li Ru-Juan Liu School of Life Science and Technology ShanghaiTech UniversityShanghai 201210China

Chemical modifications expand the composition of RNA molecules from four standard nucleosides to over 160 modified nucleosides,which greatly increase the complexity and utility of RNAs.Transfer RNAs(trnas)are the most heavily modified cellular RNA molecules and contain the largest variety of modifications.Modification of trnas is pivotal for protein synthesis and also precisely regulates the noncanonical functions of trnas.Defects in trna modifications lead to numerous human diseases.Up to now,more than 100 types of modifications have been found in trnas.Intriguingly,some modifications occur widely on all trnas,while others only occur on a subgroup of trnas or even only a specific trna.The modification frequency of each trna is approximately 7% to 25%,with 5-20 modification sites present on each trna.The occurrence and modulation of trna modifications are specifically noticeable as plenty of interplays among different sites and modifications have been discovered.In particular,trna modifications are responsive to environmental changes,indicating their dynamic and highly organized nature.In this review,we summarized the known occurrence order,cross-talk,and cooperativity of trna modifications.

关键词： trna RNA modification RNA methylation occurrence order cross-talk

Pathological significance of trna-derived small RNAs in neurological disorders

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Neural Regeneration Research 2020年第2期15卷 212-221页

作者： Chuan Qin Pei-Pei Xu Xin Zhang Chao Zhang Chang-Bin Liu De-Gang Yang Feng Gao Ming-Liang Yang Liang-Jie Du Jian-Jun Li School of Rehabilitation Medicine Capital Medical UniversityBeijingChina China Rehabilitation Science Institute BeijingChina Center of Neural Injury and Repair Beijing Institute for Brain DisordersBeijingChina Department of Spinal and Neural Functional Reconstruction China Rehabilitation Research CenterBeijingChina Beijing Key Laboratory of Neural Injury and Rehabilitation BeijingChina

Non-coding RNAs(ncRNAs) are a type of RNA that is not translated into proteins. Transfer RNAs(trnas), a type of ncRNA, are the second most abundant type of RNA in cells. Recent studies have shown that trnas can be cleaved into a heterogeneous population of ncRNAs with lengths of 18–40 nucleotides, known as trna-derived small RNAs(tsRNAs). There are two main types of tsRNA, based on their length and the number of cleavage sites that they contain: trna-derived fragments and trna-derived stress-induced RNAs. These RNA species were first considered to be byproducts of trna random cleavage. However, mounting evidence has demonstrated their critical functional roles as regulatory factors in the pathophysiological processes of various diseases, including neurological diseases. However, the underlying mechanisms by which tsRNAs affect specific cellular processes are largely unknown. Therefore, this study comprehensively summarizes the following points:(1) The biogenetics of tsRNA, including their discovery, classification, formation, and the roles of key enzymes.(2) The main biological functions of tsRNA, including its miRNA-like roles in gene expression regulation, protein translation regulation, regulation of various cellular activities, immune mediation, and response to stress.(3) The potential mechanisms of pathophysiological changes in neurological diseases that are regulated by tsRNA, including neurodegeneration and neurotrauma.(4) The identification of the functional diversity of tsRNA may provide valuable information regarding the physiological and pathophysiological mechanisms of neurological disorders, thus providing a new reference for the clinical treatment of neurological diseases. Research into tsRNAs in neurological diseases also has the following challenges: potential function and mechanism studies, how to accurately quantify expression, and the exact relationship between tsRNA and miRNA. These challenges require future research efforts.

关键词： epigenetics molecular biology neurological disorders review sequencing stress trna trna-derived fragments trna-derived small RNAs trna-derived stress-induced RNA

Apoptotic regulation and trna

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Protein & Cell 2010年第9期1卷 795-801页

作者： Yide Mei Aaron Stonestrom Ya-Ming Hou Xiaolu Yang Department of Cancer Biology and Abramson Family Cancer Research Institute University of Pennsylvania School of MedicinePhiladelphiaPA 19104USA Department of Biochemistry and Molecular Biology Thomas Jefferson UniversityPhiladelphiaPA 19107USA

Apoptotic regulation is critical to organismal homeostasis and protection against many human disease processes such as cancer.Significant research efforts over the past several decades have illuminated many signaling molecules and effecter proteins responsible for this form of programmed cell death.Recent evidence suggests that transfer RNA(trna)regulates apoptotic sensitivity at the level of cytochrome c-mediated apoptosome formation.This finding unexpectedly places trna at the nexus of cellular biosynthesis and survival.Here we review the current understanding of both the apoptotic machinery and trna biology.We describe the evidence linking trna and cytochrome c in depth,and speculate on the implications of this link in cell biology.

关键词： apoptotic regulation trna cytochrome c

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

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科学通报:英文版 2023年第18期68卷 2094-2105,M0004页

作者：黄梦涵王金涛张剑辉毛雪玲彭桂鑫林秀颖吕岱竹袁晨林桓王恩多周小龙 Key Laboratory of RNA Science and Engineering State Key Laboratory of Molecular BiologyCAS Center for Excellence in Molecular Cell ScienceShanghai Institute of Biochemistry and Cell BiologyChinese Academy of SciencesUniversity of Chinese Academy of SciencesShanghai 200031China Key Laboratory of Systems Health Science of Zhejiang Province School of Life ScienceHangzhou Institute for Advanced StudyUniversity of Chinese Academy of SciencesHangzhou 310024China School of Life Science and Technology ShanghaiTech UniversityShanghai 201210China State Key Laboratory of Marine Resource Utilization in South China Sea Hainan UniversityHaikou 570228China School of Life Sciences Hainan UniversityHaikou 570228China Analysis and Testing Center Chinese Academy of Tropical Agricultural SciencesHaikou 571101China School of Pharmacy China Pharmaceutical UniversityNanjing 211198China

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.... 详细信息

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。

关键词： trna 3-methylcytidine Methyltransferase Aminoacylation

Mitochondrial trna^(Glu) A14693G variant may modulate the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in a Han Chinese family

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Journal of Genetics and Genomics 2009年第4期36卷 241-250页

作者： Yu Ding Yongyan Li Junyan You Li Yang Bobei Chen Jianxin Lu Min-Xin Guan Zhejiang Provincial Key Laboratory of Medical Genetics School of Life Sciences Wenzhou Medical College Wenzhou 325025 China Division of Human Genetics and Center for Hearing and Deafness Research Cincinnati Children's Hospital Medical Center Cincinnati Ohio 45229 USA Department of Otolaryngology the SecondAffiliated Hospital Wenzhou Medical College Wenzhou 325000 China Deparment of Pediatrics University of Cincinnati College of Medicine Cincinnati Ohio 45229 USA

Mutations in mitochondrial 12S rRNA gene are one of the most important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we report the characterization of one Han Chinese pedigree with aminoglycoside-induced and nonsyndromic hearing loss. This Chinese family carrying the 12S rRNA A1555G mutation exhibited high penetrance and expressivity of heating impairment. In particular, penetrances of hearing loss in this family pedigree were 43.8% and 25%, respectively, when aminoglycoside-induced heating loss was included or excluded. Mutational analysis of entire mitochondrial genomes in this family showed the homoplasmic A1555G mutation and a set of variants belonging to haplogroup Y2. Of these, the A14693G variant occurred at the extremely conserved nucleotide （conventional position 54） of the TψC-loop of trna^Clu and was absent in 156 Chinese controls. Nucleotides at position 54 of trnas are often modified, thereby contributing to the structural formation and stabilization of functional trnas. Thus, the structural alteration of trna by the A14693G variant may lead to a failure in trna metabolism and impair mitochondrial protein synthesis, thereby worsening mitochondrial dysfunctions altered by the A1555G mutation. Therefore, the trna^Glu A14693G variant may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated A1555G mutation in this Chinese pedigree.

关键词： hearing loss mitochondrial DNA mutation modifier 12S rRNA trna Chinese family

ARGINYL-trna SYNTHETASE FROM Escherichia coli AFFINITY LABELING WITH 3'-OXIDIZED trna^(Arg)

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Science China Chemistry 1991年第3期34卷 297-305页

作者：程晓东林胜祥施建平王应睐 Shanghai Institute of Biochemistry Academia Sinica

The covalent modification of E. coli arginyl-trna synthetase by the 2’,3’-dialdehydederivative of trnaArg (trnaoxArg) resulted in the complete inactivation of the ATP-PPi ex-change and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gelelectrophoresis of the ArgRS-trnaoxArg covalent complexes indicated that two bands simulta-neously appeared on the gel parallel with inactivation corresponding to different higher mo-lecular weights. This result was different from that of the other aminoacyl-trna synthetaselabeling systems as previously reported. Upon the ribonuclease treatment of the modifiedArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities wererecovered. During the whole process of labeling and RNase treatment, the two activities ofthe enzyme were closely associated.

关键词： arginyl-trna synthetase arginine specific trna affinity labeling E. coli