MicroRNAs （miRNAs） control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and s...MicroRNAs （miRNAs） control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.展开更多
MicroRNAs （miRNAs） are a class of endogenous non-coding RNAs with regulatory functions. Traditionally, miRNAs are thought to play a negative regulatory role in the cytoplasm by binding to the YUTR of target genes to...MicroRNAs （miRNAs） are a class of endogenous non-coding RNAs with regulatory functions. Traditionally, miRNAs are thought to play a negative regulatory role in the cytoplasm by binding to the YUTR of target genes to degrade mRNA or inhibit translation. However, it remains a challenge to interpret the potential function of many miRNAs located in the nucleus. Recently, we reported a new type of miRNAs present in the nucleus, which can activate gene expres- sion by binding to the enhancer, and named them nuclear activating miRNAs （NamiRNAs）. The discovery of NamiRNAs showcases a complementary regulatory mechanism of miRNA, demon- strating their differential roles in the nucleus and cytoplasm. Here, we reviewed miRNAs in nucleus to better understand the function of NamiRNAs in their interactions with the enhancers. Accord- ingly, we propose a NamiRNA--enhancer-target gene activation network model to better under- stand the crosstalk between NamiRNAs and enhancers in regulating gene transcription. Moreover, we hypothesize that NamiRNAs may be involved in cell identity or cell fate determina- tion during development, although further study is needed to elucidate the underlying mechanisms in detail.展开更多
文摘MicroRNAs （miRNAs） control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.
基金supported by the National Natural Science Foundation of China (Grant No. 31671308)the Ministry of Science and Technology of China (Grant No. 2016YFC0900300)
文摘MicroRNAs （miRNAs） are a class of endogenous non-coding RNAs with regulatory functions. Traditionally, miRNAs are thought to play a negative regulatory role in the cytoplasm by binding to the YUTR of target genes to degrade mRNA or inhibit translation. However, it remains a challenge to interpret the potential function of many miRNAs located in the nucleus. Recently, we reported a new type of miRNAs present in the nucleus, which can activate gene expres- sion by binding to the enhancer, and named them nuclear activating miRNAs （NamiRNAs）. The discovery of NamiRNAs showcases a complementary regulatory mechanism of miRNA, demon- strating their differential roles in the nucleus and cytoplasm. Here, we reviewed miRNAs in nucleus to better understand the function of NamiRNAs in their interactions with the enhancers. Accord- ingly, we propose a NamiRNA--enhancer-target gene activation network model to better under- stand the crosstalk between NamiRNAs and enhancers in regulating gene transcription. Moreover, we hypothesize that NamiRNAs may be involved in cell identity or cell fate determina- tion during development, although further study is needed to elucidate the underlying mechanisms in detail.
