Functional Characteristics of a Novel Alu-originated Non-coding RNA, TIFm71

Despite Alu element represents about 10% of human genome, it was considered as "junk" for a long time. Recently, essential functions of Alu transcript have been unveiled in various biological processes. It is predicted that many noncoding RNAs have their origins within Alu element. However, functions of these Alu element-related noncoding RNAs are still elusive.;Previously, our studies identified a new CXC chemokine, named as TIF (tumor-induced factor) from xenografts that induced by mas-overexpressing CHO cells. TIF mRNA had one transcript isoform SY3 which shared identical sequence with TIF mRNA except without a 71nt-fragment located in 3'-UTR. This 71nt fragment was termed as TIFm71. Genome mapping showed that TIF gene consisted of four exons and three introns. Of interest, there was one copy of rodent Alu repeats (B1 element) in the 3'-UTR of TIF mRNA in antisense orientation and TIFm71 was part of this B1 element. It was predicted that TIFm71 itself could form a stem-loop secondary structure independently. Further in vitro study showed that TIFm71 could be released from 3'-UTR of TIF transcript in transiently transfected HEK293 cells, which implied that TIFm71 derived from Alu repeats and might function as a novel noncoding RNA. Using a GFP reporter system, it was shown that TIFm71 did not exert any significant effect on cis-gene expression.;However, a change of cell morphology from cobblestone-like to spindle-like was observed in the TIFm71 overexpressing stable cell lines, CHO-K1-M229 and Hela-M229, suggesting that cells underwent epithelial-to-mesenchymal transition (EMT). Subsequently, EMT was further conformed by changes of expression levels of EMT makers, including E-cadherin, snail and vimentin. In addition, G2/M phase arrest was also observed. It is of interest to note that the change of cell morphology became more and more drastic with the advance of cell passage. In addition, number of cells in G2/M phase was up-regulated with the increase of cell passage and more multinuclear cells appeared. These multinuclear cells gained some stem cell property as evidenced by the expression of stem cell marker CD133.;One potential signalling pathway responsible for TIFm71-induced EMT and cell cycle deregulation was ERK signalling pathway. Level of phosphorylated ERK1/2 was increased after TIFm71 overexpression. Importantly, blocking ERK signaling pathway with specific MEK inhibitor U0126, TIFm71-overexpressing stable cells returned to wild-type morphology.;Using RNA pull down assay and RNA immunoprecipitation together, it was confirmed that TIFm71 bound to non-phosphorylated ERK1/2. Besides, expression level of B1 transcript and p53 were also increased after TIFm71 overexpression.;Taken together, TIFm71 was derived from B1 repeats and functioned as a novel noncoding RNA. The stem-loop secondary structure of TIFm71 made it act as a scaffold assembling protein complex that mediates EMT and cell cycle arrest.