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The project Encyclopedia of DNA Elements (ENCODE) revealed that the mammalian genome is pervasively transcribed and many new families of RNA have been recently discovered. The majority of transcripts are long non-coding RNAs (lncRNAs) whose function is largely unknown. However, several of them may play important roles in a variety of biological processe. The recent discovery of a new class of antisense (AS) lncRNAs in the laboratory of Stefano Gusticich (SISSA, Trieste) revealed a novel regulatory mechanism of protein translation driven by lncRNAs. These molecules have been named SINEUPs, as they contain an embedded inverted SINE B2 element, required to UP-regulate translation. SINEUP translation enhancement activity has been referred to also as gene-specific ?knock-up?. Synthetic SINEUPs can be designed to redirect translation up-regulation activity to potentially any target gene of interest, thus foreseeing applications in the fields of molecular biology research, in protein manufacturing as well as in therapy of haploinsufficiencies. With this in mind, the first part of my research project was focused on exploiting SINEUP lncRNAs as a tool to increase the production of therapeutic proteins in mammalian cell factories. In this work, I could show that synthetic SINEUPs are active in Chinese Hamster Ovary (CHO) cells grown in suspension, a cell model suitable for the large-scale production of recombinant proteins. Second, I could successfully design SINEUP molecules targeting the leader sequence of secreted proteins, including monoclonal antibodies (MAbs) and cytokines. As for the few lncRNAs whose molecular mechanism has been disclosed, I believe that SINEUP activities rely on interactions with proteins. Therefore, studying the interplay between this family of lncRNA and proteins is important to uncover SINEUP function. The second part of my doctoral work was aimed at identifying the interacting proteome of SINEUP molecules, in order to shed light on the molecular mechanism governing SINEUP activity. By taking advantage of the expertise on the phage display technology available in the Laboratory of Applied Biology in Novara, I first developed a novel biotechnological pipeline for the systematic identification of protein domains that interact with a given RNA of interest (the RNA-Interacting Domainome, RIDome). Then I employed RIDome to profile the SINEUP-binding proteome. Among the top ranking interacting proteins that I have found, the interleukin enhancer binding factor 3 (ILF3) was validated in vitro and in cell-based assays. Profiling the SINEUP-bound interactome could be considered as a first step towards understanding the molecular mechanism underpinning SINEUP activity.

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of α-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. 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. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.