Engineering of a synthetic circular IRES to modulate translation of Heat-Shock Proteins mRNAs

Circular RNAs (circRNAs) are covalently closed non-coding RNAs molecules generated by alternative splicing, known as back-splicing. They have emerged as important regulators of gene expression at transcriptional, post-transcriptional and translational levels. Among their functional properties, some circRNAs harbor Internal Ribosome Entry Sites (IRESs) elements capable of promoting cap-independent translation. While IRES-mediated translation in cis is widely studied, the ability of circRNAs to regulate translation of target mRNA in trans has only recently begun to be explored. In this study, we investigated the translational regulatory role of a synthetic IRES-like element, called SynFarsa. We first characterized the ability of SynFarsa to promote cap-independent translation in cis and evaluated how sequence modifications influence its activity. Functional analysis through bicistronic luciferase including appropriate controls demonstrated that SynFarsa exhibits properties consistent with IRES-like activity. We further demonstrated that SynFarsa can be adapted to modulate translation of endogenous mRNAs in trans when guided to specific regions of targeted transcripts. In particular, SynFarsa-3’UTR, consisting of the characterized IRES-like element fused to a short reverse complement sequence base pairing a fragment of the 3’UTR, led to a significant increase in HSP90 protein levels, while HSP70 expression remained unaffected. Lastly, giving that HSP90 is known for its crucial role in regulating protein homeostasis, we examined whether SynFarsa-mediated HSP90 upregulation could affect toxic cytoplasmic FUS aggregates in the context of amyotrophic lateral sclerosis (ALS). Immunofluorescence combined with high-resolution confocal microscopy analyses were performed to assess HSP90 expression under stress conditions and its subsequent effect on FUS aggregation. Overall, this study highlights the potential of a synthetic IRES-like sequence as a translational regulatory tool and provides a framework for the development of RNA-based strategies to modulate protein expression.