An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

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.

Antisense transcription regulates the expression of sense gene via alternative polyadenylation

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.