Design of synthetic RNase-resistant RNA structures and their integration in novel riboswitch constructs to regulate mRNA stability

• Applicant: Professor Dr. Mario Mörl
• Subject Area: Biochemistry ; Bioinformatics and Theoretical Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 513266509

Flaviviruses are single-stranded RNA viruses, whose genomic RNA is flanked by heavily structured untranslated regions (UTRs). Within the 3’-UTR, evolutionarily conserved RNA elements, referred to as exoribonuclease-resistant RNAs (xrRNAs), are capable of protecting downstream regions from exonucleolytic degradation by Xrn1 and related 5’-3’-exonucleases, resulting in the production of stable decay intermediates that accumulate as long non-coding RNA (lncRNA) species in infected cells. These viral lncRNAs, also termed subgenomic flavivirus RNAs (sfRNAs), mediate pathogenicity of these viruses. We will employ bioinformatics methods to better understand the 2D and 3D structure of xrRNAs, which will enable us to design synthetic xrRNA elements with varying capacity to protect against exonucleolytic degradation. Designed elements will be studied in vivo and in vitro for their exonuclease-stalling capacity by integrating them in the 5’-UTR of reporter mRNAs whose open reading frame (ORF) is translated based on an internal ribosome entry site (IRES). The efficiency of this kind of protection will be assessed by determining mRNA half-lives. Moreover, we will employ knowledge acquired with designed xrRNAs to integrate RNA aptamer domains with compatible xrRNA structures. By combining xrRNAs with aptamers we will devise riboswitches that can form a protective structure in response to presence (or absence) of a specific ligand. These constructs will allow us to stabilize (ON switch) or destabilize (OFF switch) a specific mRNA upon ligand complexation. By using xrRNA elements that exhibit different capacities to stall exoribonucleases, this will enable us to modify the half-lives of individual transcripts. This kind of inducible regulation of RNA stability represents a novel approach in synthetic biology that allows for fine-grained modulation of both mRNAs and lncRNAs, whose half-lives are determined by Xrn1-like exoribonucleases.

• DFG Programme: Research Grants
• International Connection: Austria
• Partner Organisation: Fonds zur Förderung der wissenschaftlichen Forschung (FWF)
• Cooperation Partner: Dr. Michael Thomas Wolfinger