Small Double-stranded RNA Targeting At Promoter Activate Gene Expression By Altering The Nucleosomes Architechure

In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work in RNA interference (RNAi). Small double-stranded RNA (dsRNA) has been found to silence gene expression by an evolutionally conserved mechanism known as RNA interference or RNAi. Such dsRNAs are called small interfering RNAs or siRNA. RNAi can occur at both transcriptional and post-transcriptional levels. Surprisingly, several recent studies have found that dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa(RNA activation),and the dsRNA named saRNA(small activated RNA).SaRNA and siRNA act differently in regulation ways. SiRNA repress gene by posttranscriptional ways, and saRNA target at promoter to activate neighbour gene expression from transcriptional level. As a potential useful gene regulation way, before RNAa act as a widely used pathway in therapeutics, there are two critical questions need to resolve. The first is how to design saRNA efficiently, and the second is fully understanding the mechanism of how saRNA working on promoter.By targeting sequences in gene promoters, saRNAs readily induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa). Although previous studies put forward several principles of saRNA design, such as selecting non-CGI(CpG island, as CGI)region as target site of saRNA, but the designing of saRNA was not always effective. We speculated that TATA box and CGI are two important elements for saRNA regulation, so we divided all promoters into four types. That is type Ⅰ which have CGI and TATA box, and the TSS (transcriptional start site) on CGI; type Ⅱ,which have only CGI and the TSS on CGI; type Ⅲ,which have TATA box,but no CGI or TSS is not located in CGI, and type Ⅳ, which neither have CGI and TATA box. In order to confirm the effect of TATA box and CGI in RNAa, we choosed SOX2 gene which have type Ⅰ promoter, OCT4 and NANOG gene which have type Ⅳ promoter as the objects for screening saRNAs. Oct4,Sox2 and Nanog are the core reprogramming factors which can autoregulation by each other, that provide a possibility to detect their interaction effect after activated by saRNAs,as a strategy for assaying RNAa in functional level.When screening the saRNAs for OCT4, SOX2 and NANOG gene, we used RT-PCR, qRT-PCR and western-blot to detect the activated gene expression. After screening dsRNAs targeted promoters of OCT4,SOX2 and NANOG genes,we obtained saRNAs for all three genes. As expected, after activated by saRNA, further interaction occurred between the three factors, therefore confirmed effect of the saRNAs. In our study, we concluded that:(1) Two or three saRNAs can activate their target genes at the same time, and two saRNAs can activate same target genes synergistically; (2) Type I promoter are more effective than type IV promoter after activated by RNAa; (3) SaRNA target site closer to TSS, more upregulated expression occurred; (4) the target sites of saRNA often lies between RNA enzyme II binding sites and TSS.To study the activation mechanism of saRNA, CHIP method has been used to determine the positions of histone protein H3 and RNA polymerase II on promoter, when OCT4,SOX2 and NANOG genes were activated by designed dsRNA. The results showed that:on type I promoter,the nucleosome architecture altered completely after RNAa, however, the nucleosome architecture only altered partly after RNAa on type IV promoter,but a same rule is that nucleosomes always depleted closer to saRNA target site. In conclusion, saRNA induce nucleosomes reposition, then modify the architecture and result in transcriptional activation. However, nucteosomes reposition just determine turn-on or turn-off of transcription, the activated transcriptional efficiency was determined by cooperation between transcriptional regulatory elements and saRNA targets location. For type I promoter, the transcriptonal regulatory elements including TATA box and CGI, and for type IV, saRNA target located in enhancer and proximal promoter may be easy to improve transcriptional efficiency.We concluded that, when dsRNA binds to a particular sequence downstream TATA box, or binds to transcription factor binding preparation areas such as proximal promoter and enhancer, this DNA/RNA motif began to recruit chromatin remodeling complex, such as RNA polymerase II and other transcriptional factors. Then results in nucleosome remodeling and nucleosome depleted region (NDR) produce at the closer region of saRNA target site and recruiting PIC later. IfNDR locates at the downstream of dsRNA target site, the PIC access to TSS become smoothly and make transcription activated. If saRNA target in enhancer, the NDR could make enhancer exposed to induce saRNA/DNA motif recruit transcriptional proteins to activate transactivation.Overall, using cooperation of transcriptional regulatory elements and reasonable target site is an ideal method to design more effective saRNA, which aimed at activating gene with different type of promoter.