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
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
自然反义转录物(natural antisense transcript,NAT)是一种在原核和真核生物中具有重要基因表达调控功能的非编码RNA分子。NAT通常与其靶分子通过序列互补形成自然有义-反义转录物(natural sense-antisense transcript,NSAT)配对的双链R...自然反义转录物(natural antisense transcript,NAT)是一种在原核和真核生物中具有重要基因表达调控功能的非编码RNA分子。NAT通常与其靶分子通过序列互补形成自然有义-反义转录物(natural sense-antisense transcript,NSAT)配对的双链R N A,引起靶分子的降解或翻译抑制。鉴于非编码R N A介导的基因调控生物学事件的普遍性,以及最近各种生物的大规模基因组测序的完成,对N A T进行高通量的筛选与深入研究显得尤为重要和迫切。展开更多
Trans-acting small interfering RNAs （ta-siRNAs） and natural cis-antisense siRNAs （cisnat-siRNAs） are recently discovered small RNAs （sRNAs） involved in post-transcriptional gene silencing, ta-siRNAs are transcri...Trans-acting small interfering RNAs （ta-siRNAs） and natural cis-antisense siRNAs （cisnat-siRNAs） are recently discovered small RNAs （sRNAs） involved in post-transcriptional gene silencing, ta-siRNAs are transcribed from genomic loci and require processing by microRNAs （miRNAs）. cis-nat-siRNAs are derived from antisense RNAs produced by the simultaneous tran- scription of overlapping antisense genes. Their roles in many plant processes, including pathogen response, are mostly unknown. In this work, we employed a bioinformatic approach to identify ta-siRNAs and cis-nat-siRNAs in cassava from two sRNA libraries, one constructed from healthy cassava plants and one from plants inoculated with the bacterium Xanthomonas axonopodis pv. manihotis （Xam）. A total of 54 possible ta-siRNA loci were identified in cassava, including a homo- log of TAS3, the best studied plant ta-siRNA. Fifteen of these loci were induced, while 39 were repressed in response to Xam infection. In addition, 15 possible c/s-natural antisense transcript （cis-NAT） loci producing siRNAs were identified from overlapping antisense regions in the genome, and were found to be differentially expressed upon Xam infection. Roles of sRNAs were predicted by sequence complementarity and our results showed that many sRNAs identified in this work might be directed against various transcription factors. This work represents a significant step toward understanding the roles of sRNAs in the immune response of cassava.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 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.
文摘自然反义转录物(natural antisense transcript,NAT)是一种在原核和真核生物中具有重要基因表达调控功能的非编码RNA分子。NAT通常与其靶分子通过序列互补形成自然有义-反义转录物(natural sense-antisense transcript,NSAT)配对的双链R N A,引起靶分子的降解或翻译抑制。鉴于非编码R N A介导的基因调控生物学事件的普遍性,以及最近各种生物的大规模基因组测序的完成,对N A T进行高通量的筛选与深入研究显得尤为重要和迫切。
基金funded by Dirección de Investigaciones Sede Bogotá(Grant No.201010011438)Colciencias(Contract No. 221-2008)
文摘Trans-acting small interfering RNAs （ta-siRNAs） and natural cis-antisense siRNAs （cisnat-siRNAs） are recently discovered small RNAs （sRNAs） involved in post-transcriptional gene silencing, ta-siRNAs are transcribed from genomic loci and require processing by microRNAs （miRNAs）. cis-nat-siRNAs are derived from antisense RNAs produced by the simultaneous tran- scription of overlapping antisense genes. Their roles in many plant processes, including pathogen response, are mostly unknown. In this work, we employed a bioinformatic approach to identify ta-siRNAs and cis-nat-siRNAs in cassava from two sRNA libraries, one constructed from healthy cassava plants and one from plants inoculated with the bacterium Xanthomonas axonopodis pv. manihotis （Xam）. A total of 54 possible ta-siRNA loci were identified in cassava, including a homo- log of TAS3, the best studied plant ta-siRNA. Fifteen of these loci were induced, while 39 were repressed in response to Xam infection. In addition, 15 possible c/s-natural antisense transcript （cis-NAT） loci producing siRNAs were identified from overlapping antisense regions in the genome, and were found to be differentially expressed upon Xam infection. Roles of sRNAs were predicted by sequence complementarity and our results showed that many sRNAs identified in this work might be directed against various transcription factors. This work represents a significant step toward understanding the roles of sRNAs in the immune response of cassava.
