H-Ras Pre-mRNA Contains A Regulatory Non-coding RNA

Non-coding RNAs (ncRNAs) have emerged as one of the most abundant regulatory molecules. However, their roles and functions are significantly different from those of proteins. Moreover, around 95% of the human genome contains non-coding DNA. ncRNAs contribute by far the majority of human transcriptional units, and the functions of the most are yet unknown. Here, we highlight that an important RNA sequence region, encompassing an exon-intron hairpin loop (also called IDX-rasISS1), of the H-Ras pre-mRNA may encode an ncRNA that regulates p68 RNA helicase. This observation is remarkable owing to the fact that this helicase is responsible for upregulating the hairpin loop. This indicates that an inhibitory feedback mechanism acting on the p68 RNA helicase is mediated by higher structural levels of the hairpin-loop. Initially, two observations prompted the present study: i) previous results revealed down-regulation of p68 RNA helicase resulting from overexpression of the IDX hairpin loop in HeLa cells, and ii) the secondary structure of the IDX hairpin loop resembles pri-miRNAs, implying that an miRNA could be processed from the hairpin loop-containing pri-miRNA and regulate 68 RNA helicase. To validate our hypothesis, we directly compared p68 RNA sequences and the hairpin loop in silico. Furthermore, RNAi assays containing the hairpin loop as an miRNA precursor were conducted, using the pTer vector, to explore the effects on 68 RNA helicase expression levels. These RNAi analyses were quantified by Western blots (using anti-p68 RNA helicase and anti-EIF2α) and Fluc/Rluc 3’UTRs/CDS assays. The effect of hairpin loop overexpression on cell growth and cancer processes was also investigated by analyzing cell-cycle phases and miR-206 expression. Finally, alternative splicing microarrays containing apoptosis targets were incubated to verify whether pre-mRNAs other than H-Ras could also present a similar hairpin loop structure regulated by p68 RNA helicase. We observed that overexpression of the hairpin loop does not activate the phosphorylation of EIF2α and, therefore, does not activate PKR interferon-induced apoptosis. Moreover, a similar effect on p68 RNA helicase-mediated interference is observed during the upregulation of the hairpin loop. Finally, we also identified a similar hairpin loop-like structure in an alternative splicing region of MAPK12/ERK6. Thus it can be inferred from our findings that the alternative splicing exon IDX from H-Ras, coupled with the immediately downstream intron sequences, may contain an ncRNA. We also unveil one potential function of this ncRNA whose expression is regulated by alternative splicing decisions.