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
Nitric oxide(NO)is a crucial signaling molecule that regulates a wide range of metabolic pathways in different strata of organisms.In plants,nitrate reductase(NR)is a key enzyme for NO biosynthesis.There are two NR-en...Nitric oxide(NO)is a crucial signaling molecule that regulates a wide range of metabolic pathways in different strata of organisms.In plants,nitrate reductase(NR)is a key enzyme for NO biosynthesis.There are two NR-encoding genes in Arabidopsis genome,NIA1 and NIA2,which are precisely regulated and expressed in a tissue-specific manner.In this study,we found that the natural antisense transcript as-NIA1,transcribed from the 30 UTR of NIA1,stabilizes NIA1 mRNA to maintain its circadian oscillation in plants grown under the light/dark cycle.Importantly,as-NIA1-dependent NIA1 mRNA stability is indispensable for NIA1-mediated NO biosynthesis in guard cells and natural stomatal closure.Moreover,we revealed that polypyrimidine tract-binding 3(PTB3)regulates the stabilization of NIA1 mRNA by directly binding to UC-rich elements of as-NIA1.We further found that MTA deposits N^(6)-methyladenosine(m6 A)on as-NIA1,facilitating the as-NIA1-PTB3 interaction in vivo,in agreement with RNA structure prediction in that m6 Amediated structural alterations expose the UC-rich elements to enhance the accessibility of PTB3.Taken together,these findings reveal a novel molecular mechanism by which plants precisely manipulate NO biosynthesis to modulate light/dark-regulated stomatal movement,highlighting the coupling of RNA epigenetic modifications and structures shaping RNA-protein interactions in the regulation of hormone biosynthesis.展开更多
Non-coding RNAs(nc RNAs),such as micro RNAs and large intergenic non-coding RNAs,have been shown to play essential roles in regulating pluripotency.Yet,it is not clear the role of natural antisense transcripts(NATs),a...Non-coding RNAs(nc RNAs),such as micro RNAs and large intergenic non-coding RNAs,have been shown to play essential roles in regulating pluripotency.Yet,it is not clear the role of natural antisense transcripts(NATs),also belonging to nc RNAs,in embryonic stem cells.However,the role of NATs in embryonic stem cells remains unknown.We further confirmed the expression of the NATs of three key pluripotency genes,Oct4,Nanog and Sox2.Moreover,overexpression of Sox2-NAT reduces the expression of Sox2 protein,and slightly enhances the Sox2 m RNA level.Altogether,our data indicated that like other nc RNAs,NATs might be involved in pluripotency maintenance.展开更多
Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological proce...Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense （S-AS） gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA （pA） site in sense gene RNASEH2C, which generated longer 3＇ untranslated region （3＇UTR） and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene＇s protein level in RNASEH2C.KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.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funded by the National Key R&D Program of China 2022YFD1400800the National Science Foundation of China,China(no.32170293).
文摘Nitric oxide(NO)is a crucial signaling molecule that regulates a wide range of metabolic pathways in different strata of organisms.In plants,nitrate reductase(NR)is a key enzyme for NO biosynthesis.There are two NR-encoding genes in Arabidopsis genome,NIA1 and NIA2,which are precisely regulated and expressed in a tissue-specific manner.In this study,we found that the natural antisense transcript as-NIA1,transcribed from the 30 UTR of NIA1,stabilizes NIA1 mRNA to maintain its circadian oscillation in plants grown under the light/dark cycle.Importantly,as-NIA1-dependent NIA1 mRNA stability is indispensable for NIA1-mediated NO biosynthesis in guard cells and natural stomatal closure.Moreover,we revealed that polypyrimidine tract-binding 3(PTB3)regulates the stabilization of NIA1 mRNA by directly binding to UC-rich elements of as-NIA1.We further found that MTA deposits N^(6)-methyladenosine(m6 A)on as-NIA1,facilitating the as-NIA1-PTB3 interaction in vivo,in agreement with RNA structure prediction in that m6 Amediated structural alterations expose the UC-rich elements to enhance the accessibility of PTB3.Taken together,these findings reveal a novel molecular mechanism by which plants precisely manipulate NO biosynthesis to modulate light/dark-regulated stomatal movement,highlighting the coupling of RNA epigenetic modifications and structures shaping RNA-protein interactions in the regulation of hormone biosynthesis.
基金supported by the National Natural Science Foundation of China(31271547)the Natural Science Foundation of Tianjin,China(14JYBJC23600)+3 种基金the National Key Basic Research and Development Program of China(2010CB833603)the Program for New Century Excellent Talents(NCET-13-0293)the 111 Project Grant(B08011)the Funds for National Basic Science Personnel Training(J1103503)
文摘Non-coding RNAs(nc RNAs),such as micro RNAs and large intergenic non-coding RNAs,have been shown to play essential roles in regulating pluripotency.Yet,it is not clear the role of natural antisense transcripts(NATs),also belonging to nc RNAs,in embryonic stem cells.However,the role of NATs in embryonic stem cells remains unknown.We further confirmed the expression of the NATs of three key pluripotency genes,Oct4,Nanog and Sox2.Moreover,overexpression of Sox2-NAT reduces the expression of Sox2 protein,and slightly enhances the Sox2 m RNA level.Altogether,our data indicated that like other nc RNAs,NATs might be involved in pluripotency maintenance.
基金We thank Drs. Li Jin, Feng Qian, Jun Zhu and Hongjie Yao for constructive suggestions of this manuscript. The vectors of CRISPR/ Cas9 are the generous gifts from Drs. Yangming Wang and Yong- ming Wang. This work was supported by the National Basic Research Program （973 Program） （Nos. 2013CB530700 and 2015CB943000 to T. N.） and National Natural Science Foundation of China （Grant Nos. 31471192 and 31521003 to T. N.）.
文摘Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense （S-AS） gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA （pA） site in sense gene RNASEH2C, which generated longer 3＇ untranslated region （3＇UTR） and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene＇s protein level in RNASEH2C.KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.
