SINEUPs, a new class of antisense long non-coding RNAs that enhance synthesis of target proteins in cells: molecular mechanisms and applications

Thanks to continuous advances in sequencing technologies, we know that a huge number of non-coding RNAs are transcribed from mammalian genomes. Of these, long non-coding RNAs (lncRNAs) represent the widest and most heterogeneous class. An increasing number of studies are unveiling lncRNA functions, supporting their active role in regulating gene expression. Regardless of lncRNAs specific functional features, their organization into discrete domains seems to represent a common denominator. Through such domains lncRNAs can recruit and coordinate the activity of multiple effectors, thus working as “flexible modular scaffolds”. This model has globally driven towards the quest for regulatory elements within lncRNAs, with a special attention on functional cues deriving from RNA folding. Since transposable elements (TEs) represent 40% of nucleotides of lncRNA sequences, they have been proposed as candidate functional modules. Carrieri and colleagues recently reported that an embedded inverted SINEB2 element acts as a functional domain in antisense (AS) Uchl1, an AS lncRNA able to increase translation of partially-overlapping protein-coding sense Uchl1 mRNA. AS Uchl1 regulatory properties depend on two RNA domains. A 5' overlapping sequence to the sense transcript is the Binding Domain (BD) and drives specificity of action. An embedded inverted SINEB2 element functions as Effector Domain (ED) conferring translational activation power. AS Uchl1 is the representative member of a new class of lncRNAs, named SINEUPs, as they rely on a SINEB2 element to UP-regulate translation. AS Uchl1 activity can be transferred to a synthetic construct by manipulating the AS sequence in the BD, suggesting the potential use of AS Uchl1- derived synthetic SINEUPs as tools to increase translation of selected targets. This work was the first example of a specific biological function assigned to an embedded TE leading to the hypothesis that embedded TEs provide functional modules to lncRNAs. A major limit to the application of SINEUPs is represented by the poor knowledge of the basic mechanisms underlying the biological activity of the ED. A crucial challenge becomes the identification of secondary structures that may confer characteristic protein binding properties. Protein partners would modulate SINEUPs action and contribute to achieve specific functional outputs. In this thesis, I focus on understanding the molecular basis of SINEUPs activity in cells and I discuss the potential applications of synthetic SINEUPs as translation enhancers. First, I investigated the structural basis for translation activation mediated by the ED of SINEUPs. I pointed out that specific structural regions, containing a short terminal hairpin, are involved in the ability of natural and synthetic SINEUPs to increase translation of target mRNAs. Next, I identified protein partners modulating the activity of SINEUPs in cells. I found that AS Uchl1 interacts with the interleukin enhancer-binding factor 3 (ILF3) and that the presence of the inverted SINEB2 favors binding in vivo. In particular, I demonstrated that the AS Uchl1-embedded TEs, inverted SINEB2 and Alu, direct AS Uchl1 localization to ILF3-containing complexes, thus contributing to AS Uchl1 bias towards nuclear localization. I thus suggest that nuclear retention could represent a possible mechanism regulating SINEUP activity. I also validated the scalability of synthetic SINEUPs as tools to increase protein synthesis of targets of choice. I showed that SINEUP technology can be adapted to a broader number of targets, with interesting potential applications in different fields, from biotechnology to therapy. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Their modular structure can be exploited to successfully design synthetic SINEUPs against selected endogenous targets, supporting their efficacy as tools to modulate gene expression in vitro and in vivo. Hence, I propose SINEUPs as versatile tools to enhance translation of mRNAs of choice.