RNA-mediated targeting and interactomics

RNA-mediated approach for homogeneous and tunable intracellular protein expression. Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product in eukaryotic cells. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. We have developed an RNA-based electroporation method, which offers extremely high transfection efficiency.  Protein expression takes place few hours post-transfection, lasts at least for 48 h, and occurs evenly across cells, with a transfection efficiency reaching up to 98% cells in most cell lines tested. Most interestingly, the level of protein expressed can be finely tuned by simply modulating the amounts of mRNAs transfected. We further develop this methodology by improving the stability of the mRNAs using NTP derivatives and by optimizing the 5’ and 3’ UTR regions. mRNA electrotransfer could supplant the conventional DNA-based transient expression system.
Development of the Holdup assay for detection and affinity quantification of RNA-protein complexes and application to ncRNAs.  While 70–80% of the human genome is transcribed, only 2% of it encodes proteins. The vast majority of the human transcriptome consists of noncoding RNAs (ncRNAs). The networks in which ncRNAs act as key regulators influence a wide range of targets to control specific cell biological responses. Of particular relevance, ncRNAs have been identified as oncogenic drivers and tumour suppressors in all major cancer types. Two groups of ncRNAs have been characterized, the small and long ncRNA (sncRNAs and lncRNAs, respectively). Small ncRNAs (sncRNAs), which include among others micro-RNAs, small interference RNAs and piwi-interacting RNAs, have been extensively studied. In contrast, much less is known about the function of long noncoding RNAs (lncRNAs). Therefore, we aim to understand the complex networks of interactions that lncRNAs coordinate in the cellular context. To this end, we are developing a new variation of the Holdup assay to unravel the quantitative protein interactome of selected lncRNA molecules. As a first model, we will study the interactome of the Damage Induced NOncoding lncRNA, DINO. DINO has been characterized as a TP53 transcriptional target and functional modulator that causes TP53 reactivation in HPV-positive cervical cancer cells. The DINO lncRNA interactome may shed light on its potential role in the development of HPV-positive cancers.