Insights on the molecular mechanisms of SINEUP activity

Translation initiation during protein synthesis is one of the rate limiting steps in regulating gene expression in eukaryotes. It may occur through a cap- dependent or independent mechanism. Cap-independent translation initiation usually takes place when the canonical cap-dependent process involving the cap-eIF4F complex and/or the ternary complex (TC) are inhibited, mostly during cellular stress. It can be activated by modular RNA elements like internal ribosome entering sites (IRESs) that can act alone or in conjunction with other RNA cis-acting elements such as upstream open reading frames (uORF), terminal oligopyrimidines (TOPs) and N6-methyladenosine (m6A). Recently, antisense (AS) long non-coding RNAs (lncRNAs) to mouse and human gene targets were reported to up-regulate translation of those genes, representing a new functional class of lncRNAs termed SINEUPs, for SINE sequences UP-regulating translation. SINEUP modular organization consists of a binding domain (BD) which specifically targets the mRNA of interest by an antisense sequence to its 5’UTR, and an effector domain (ED) constituted by an embedded SINE sequence which confers the biological function of translation activation. However, the underlying molecular mechanism mediated by SINEUPs is unknown. Here, I show using both SINEUP- and IRES-activity assays that the ED of AS Uchl1, the lncRNA representative member of SINEUPs, promotes cap-independent translation through an internal ribosome entry site that is mediated by the inverted SINEB2 stem loop 1 (SL1) hairpin structure, which probably recruits the ribosome using an IRES-like mechanism similar to the one in Hepatitis Virus C. Since synthetic SINEUPs can be targeted to potentially any gene of interest by swapping the BDs, they can be used as molecular tools, in protein manufacturing and RNA therapeutics to increase protein synthesis. Therefore, in this work I optimized a miniaturized version, the synthetic microSINEUP, and showed for the first time that SINEUPs are functional in Drosophila cells. In addition, taking advantage of SINEUP modular organization, I synthesized IRES-containing SINEUPs, called SINEUP-IRESs, that present cellular and viral IRESs acting as ED in SINEUP RNA molecules and therefore proving that IRES sequences can activate translation in trans through a BD. By the study of IRES-containing c-myc mRNA, I discovered the ability of natural IRES elements to increase endogenously expressed, targeted proteins synthesis in trans. These data are the first evidence for a potential new molecular mechanism of gene expression control with long-range consequences in health and disease.