Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

Binding of microRNAs (miRNA) to mRNAs normally results in post-transcriptional repression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (crosslinking, ligation, and sequencing of miRNA-target RNA hybrids) data, and identified numerous candidate TDMD triggers based on their ability to induce non-templated nucleotide addition at the miRNA 3 end. When overexpressed in HEK293T cells, nine highly conserved triggers induce degradation of corresponding miRNAs. Consistent with previous reports, both the TDMD base-pairing and surrounding sequences are important for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduce miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes an apoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated that they can functionally cooperate with the encoded proteins. HEK 293T cells were transfected with plasmids encoding a GFP-reporter containing either a BCL2L11 or TRIM9 TDMD trigger in the 3'UTR. Total RNAs were extracted from transfected cells and subjected to Poly-A RNA sequencing and small RNA sequencing. This experiment was designed to assess the influence of TDMD triggers on miRNAs. TDMD stands for "target RNA directed microRNA degradation", in which extensive base-pairing between target RNA and miRNA leads to miRNA degradation. Here, we examine whether expression of TDMD triggers from BCL2L11 or TRIM9 could degrade their corresponding miRNAs ( miR-221/222 or miR-218, respectively). After transfection of plasmids expressing TDMD-triggers, HEK293T cell were lyszed by Trizol for total RNA extraction. Poly-A RNAs were selected by oligo-dT beads and 18-40nt RNAs were selected while constructing NEBNext small RNA seq library, and subjected to Illumina sequencing. Two biological replicateswere performed for each TDMD trigger.