SINEUPS are antisense long non-coding RNAS that increase synthesis of target proteins in cells: applications in biotechnology

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.