Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic proce...Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic process of RNA export in live cells is challenging.When dye-labeled RNA enters the cytoplasm,the dye molecules are released upon degradation of the RNA,allowing them to re-enter the cell nucleus.As a result,the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined.To address this common limitation,we report the design of a smart probe that can only check into the nucleus once.When adding to cells,this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H_(2)S.This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization.After disassociating from exported RNAs in cytoplasm,the probe preferentially enters lysosomes rather than cell nucleus,enabling real-time quantitative measurement of nuclear RNA exports.Using this probe,we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells.Interestingly,we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.展开更多
AIM: To search for the presence of cis elements in hepatitis D virus(HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferas...AIM: To search for the presence of cis elements in hepatitis D virus(HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid p DM138. Twenty c DNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pD M138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by realtime polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter m RNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.展开更多
生物体中遗传信息的传递遵循中心法则,即从基因组DNA传递到RNA,再进一步传递到蛋白质。作为遗传信息传递的中间一环,RNA需要在细胞核中生成,并进一步被转运到细胞质中,其中涉及的RNA出核转运过程则是保证遗传信息精确传递的重要步骤,并...生物体中遗传信息的传递遵循中心法则,即从基因组DNA传递到RNA,再进一步传递到蛋白质。作为遗传信息传递的中间一环,RNA需要在细胞核中生成,并进一步被转运到细胞质中,其中涉及的RNA出核转运过程则是保证遗传信息精确传递的重要步骤,并参与了基因表达的精确调控过程。绝大多数的mRNA通常在细胞核内包装形成mRNP,并在TREX复合体(transcription-export complex,TREX)和出核因子NXF1(nuclear RNA export factor 1)的帮助下转运出核。另有一小部分的mRNA则以CRM1(chromosome region maintenance protein 1)依赖的方式进行出核转运。大量研究表明,mRNA出核过程不仅与上游的转录和加工相偶联,而且能够反向调控上游步骤。此外,细胞核中RNA的出核机器与降解机器相互竞争,共同决定新生RNA的核内命运。尤为重要的是,当mRNA出核受阻时,在细胞和个体层面上会产生不同程度的生理或病理缺陷。最新的研究表明,除mRNA以外,circRNA和lncRNA等其他RNA分子也被认为能够转运出核并翻译产生蛋白质,参与各类生物学过程的调控,其出核过程则涉及一些新的受体因子和调控蛋白。本文将结合最新的研究进展,对已知RNA出核转运通路的相关具体过程和调控机制进行总结,并对领域内后续研究的重难点进行讨论和展望。展开更多
基金supported by the National Natural Science Foundation of China(Nos.21775001,21874137,21974001,21976183and 22074001)Natural Science Foundation of Anhui Province(No.1808085MB32)Nature and Science Foundation from Anhui Province Ministry of Education(No.KJ2019A0011)。
文摘Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic process of RNA export in live cells is challenging.When dye-labeled RNA enters the cytoplasm,the dye molecules are released upon degradation of the RNA,allowing them to re-enter the cell nucleus.As a result,the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined.To address this common limitation,we report the design of a smart probe that can only check into the nucleus once.When adding to cells,this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H_(2)S.This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization.After disassociating from exported RNAs in cytoplasm,the probe preferentially enters lysosomes rather than cell nucleus,enabling real-time quantitative measurement of nuclear RNA exports.Using this probe,we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells.Interestingly,we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.
基金Supported by A grant from Fundao para a Ciência e Tecnologia,Portugal to Freitas N
文摘AIM: To search for the presence of cis elements in hepatitis D virus(HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid p DM138. Twenty c DNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pD M138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by realtime polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter m RNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.
文摘生物体中遗传信息的传递遵循中心法则,即从基因组DNA传递到RNA,再进一步传递到蛋白质。作为遗传信息传递的中间一环,RNA需要在细胞核中生成,并进一步被转运到细胞质中,其中涉及的RNA出核转运过程则是保证遗传信息精确传递的重要步骤,并参与了基因表达的精确调控过程。绝大多数的mRNA通常在细胞核内包装形成mRNP,并在TREX复合体(transcription-export complex,TREX)和出核因子NXF1(nuclear RNA export factor 1)的帮助下转运出核。另有一小部分的mRNA则以CRM1(chromosome region maintenance protein 1)依赖的方式进行出核转运。大量研究表明,mRNA出核过程不仅与上游的转录和加工相偶联,而且能够反向调控上游步骤。此外,细胞核中RNA的出核机器与降解机器相互竞争,共同决定新生RNA的核内命运。尤为重要的是,当mRNA出核受阻时,在细胞和个体层面上会产生不同程度的生理或病理缺陷。最新的研究表明,除mRNA以外,circRNA和lncRNA等其他RNA分子也被认为能够转运出核并翻译产生蛋白质,参与各类生物学过程的调控,其出核过程则涉及一些新的受体因子和调控蛋白。本文将结合最新的研究进展,对已知RNA出核转运通路的相关具体过程和调控机制进行总结,并对领域内后续研究的重难点进行讨论和展望。
