A widespread repression of miRNA has been described in almost every type of cancer, caused by promoter silencing, transcriptional regulation or alterations in miRNA processing. Our hypothesis is that also altered miRNAs degradation contributes to miRNA repression in cancer. This idea was suggested by recent data showing that miRNAs can be degraded upon interaction with their targets, if the targets fulfill precise requirements in terms of complementarity. We developed a way to circumvent this problem by exploiting 4S-Uridine (4SU), a low-frequency naturally occurring modified base that can be incorporated in place of Uridine into the nascent RNA. 4SU serves as an attachment point for a thiol-specific biotinylation that allow the separation of newly transcribed RNA from the total RNA population. This method was originally developed to study the dynamics of transcription and decay of mRNAs. We adapted the protocol to microRNAs: 4SU is added to the medium for a period of time sufficient to label also mature microRNAs (3hrs), then 4SU is removed from the medium and the decay of labeled species is monitored, at different time points, during the chase period. We can isolate enough materials to generate high-throughput sequencing libraries, thus we can simultaneously analyze the decay of all expressed miRNAs and their 3’ modified forms (“isomiRs”, that can display differential stability compared to canonical miRNAs). We applied this approach to analyze the dynamics of miRNA decay under physiological conditions (growing vs. quiescent cells) and under oncogenic stimuli.
In this project, I propose to rule out the hypothesis that endogenous RNA targets could regulate miRNAs by a target-induced miRNA decay (TIMD) mechanism. By a rigorous pipeline that combines a series of bioinformatics and experimental analyses, I will identify and validate a group of endogenous targets involved in TIMD.To explicitly validate this hypothesis, our team will exploit CRISPR/Cas9 gene editing to delete miRNA-responsive elements (MREs) from the 3'UTR of candidate transcripts, to specifically interfere with miRNA:target interaction while keeping mRNA and miRNA expression at their endogenous levels (WP1). State-of-the-art quantitation of gene and miRNA expression by digital PCR and RNA-sequencing will be used to measure miRNA and target expression upon manipulation. Additional tools specifically developed in our lab, namely IsomiRage (for the analysis of miRNA isoforms by small-RNA sequencing) and 4SU-labelling (to monitor miRNA decay), will be also exploited to achieve a comprehensive characterization of the bidirectional effects associated with target:miRNA interaction.