NEW FINDINGS INTO TARGET-DIRECTED MICRORNA DEGRADATION MECHANISM

MiRNAs are a class of small non-coding RNAs that function in post-transcriptional gene silencing by interacting with target RNAs. A novel mechanism in control of miRNA levels, called Target-Directed miRNA degradation (TDMD), has been described, which involves specific transcripts able to interact with miRNAs and induce miRNA degradation. We showed that an endogenous RNA transcript, Serpine1, uses TDMD to control levels and activity of miR:30b/c in murine fibroblasts. It is unknown how many other TDMD transcripts exist; therefore, we developed TDMDfinder, a computational pipeline and free webtool, that identifies “high confidence” TDMD interactions in the Human and Mouse transcriptomes by combining sequence alignment and feature selection approaches. Our predictions suggested that TDMD is widespread, with potentially every miRNA controlled by endogenous targets. We experimentally tested 37 TDMDfinder predictions, of which 17 showed TDMD effects as measured by RT-qPCR and small RNA sequencing, linking the miR-17, miR-19, miR-30, miR-221, miR-26 and miR-23 families to novel endogenous TDMD triggers. Computational analyses performed using the multiomic TCGA platform substantiated the possible involvement of many TDMD transcripts in human cancer and highlighted 36 highly significant pan-cancer interactions, suggesting TDMD as a new potential oncogenic mechanism. Focusing on the SERPINE1:miR30b/c pair as a model, we applied molecular and genetic approaches to manipulate TDMD and investigate the effects afforded by miRNA degradation in breast cancer. Our results suggested that TDMD is used by breast cancer cells to keep low miRNA activity and provide a selective advantage in various cancer phenotypes. In order to identify all TDMD-regulated miRNAs in breast cancer, we inhibited the TDMD mechanism by knocking down a critical player of the pathway, the ZSWIM8 culling-RING ubiquitin ligase substrate adapter. We repressed ZSWIM8 by CRISPR interference in a set of 8 different breast cancer (BC) cell lines, representing the entire spectrum of BC subtypes. Our analyses showed that loss of ZSWIM8 caused significantly increased accumulation of 31 miRNAs, without any evidence of transcriptional regulation, thus expanding the role of endogenous TDMD in sculpting miRNA levels. Finally, to identify the TDMD triggers in BC, we designed a strategy based on the combined use of TDMDfinder predictions and experimental CRISPR-based tools. We performed a proof-of-concept validation of the approach by identifying the endogenous TDMD trigger (NREP) for miR-29b-3p in SUM159PT and BT549, proving that this can be an effective strategy to quickly identify TDMD substrates and triggers.