High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many ant...High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.展开更多
Synthetic RNA-based switches provide distinctive merits in modulating gene expression.Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation,paving the way for innovative tools t...Synthetic RNA-based switches provide distinctive merits in modulating gene expression.Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation,paving the way for innovative tools that can sense and manipulate cellular processes.In this research,we have developed programmable ribozymes that are capable of suppressing gene expression in response to specific,endogenously expressed trigger RNAs.We engineer ribozymes by introducing upstream antisense sequences(anti-ribozymes)to inhibit the self-cleaving activity of the hammerhead ribozyme and open the expression of the target gene.The trigger RNA is designed to recognize and bind to complementary sequences within the anti-ribozymes,thereby inhibiting their ability to direct protein synthesis.The anti-ribozyme performance is optimized by regulating the essential sequence modules that play a crucial role in determining the specificity and efficiency of the anti-ribozyme’s interaction with its trigger RNA.By applying this switch mechanism to various ribozyme designs,we have shown that it is possible to achieve control over gene expression across a wide range of trigger RNAs.By exploiting these pro-grammable anti-ribozymes,we aim to create a powerful tool for controlling gene expression in mammalian cells,which could have important implications for basic research,disease diagnosis,and therapeutic interventions.展开更多
基金supported by the National Natural Science Foundation of China(31301958)the Chinese Postdoctoral Science Foundation(2013T60808)
文摘High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.
基金supported by a grant from the Ministry of Science and Technology of the People’s Republic of China(Grant No.2021YFC2101700 and Grant No.2018YFA0900100)National Key Research and Development Program of China(Grant No.2021YFA0910700)+1 种基金the Chinese Academy of Sciences[No.XDB0480100 of the Strategic Priority Research Program]and CAS Youth Interdisciplinary TeamThis work was also supported by Vanke Special Fund for Public Health and Health Discipline Development,Tsinghua University(2022Z82WKJ006).
文摘Synthetic RNA-based switches provide distinctive merits in modulating gene expression.Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation,paving the way for innovative tools that can sense and manipulate cellular processes.In this research,we have developed programmable ribozymes that are capable of suppressing gene expression in response to specific,endogenously expressed trigger RNAs.We engineer ribozymes by introducing upstream antisense sequences(anti-ribozymes)to inhibit the self-cleaving activity of the hammerhead ribozyme and open the expression of the target gene.The trigger RNA is designed to recognize and bind to complementary sequences within the anti-ribozymes,thereby inhibiting their ability to direct protein synthesis.The anti-ribozyme performance is optimized by regulating the essential sequence modules that play a crucial role in determining the specificity and efficiency of the anti-ribozyme’s interaction with its trigger RNA.By applying this switch mechanism to various ribozyme designs,we have shown that it is possible to achieve control over gene expression across a wide range of trigger RNAs.By exploiting these pro-grammable anti-ribozymes,we aim to create a powerful tool for controlling gene expression in mammalian cells,which could have important implications for basic research,disease diagnosis,and therapeutic interventions.
