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Updated: Oct 12, 2025
Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs
Published on: June 12, 2018
Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins
Lu Li1,2, Peike Sheng1,2, Tianqi Li1,2
1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Abstract:
Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. 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 (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.
Insights
Extensive base-pairing can trigger microRNA (miRNA) degradation, a process known as target RNA-directed miRNA degradation (TDMD). Researchers identified widespread TDMD triggers in target RNAs, revealing a new layer of gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) typically repress gene expression post-transcriptionally.
- Extensive miRNA-target RNA base-pairing can lead to miRNA degradation (TDMD).
Purpose of the Study:
- To systematically identify target RNAs that trigger miRNA degradation (TDMD).
- To investigate the mechanism and functional consequences of TDMD.
Main Methods:
- Analysis of Argonaute-CLASH data to identify miRNA-target RNA interactions.
- Functional validation of candidate TDMD triggers in cell lines.
- CRISPR-Cas9 gene editing to assess the role of endogenous triggers and ZSWIM8.
Main Results:
- Identified numerous candidate TDMD triggers capable of inducing nontemplated nucleotide addition at the miRNA 3' end.
- Eight candidate triggers were confirmed to induce degradation of specific miRNAs.
- Both base-pairing and flanking sequences are crucial for TDMD.
- Knockout of triggers or ZSWIM8 reduced miRNA degradation.
- Degradation of miR-221/222 by a BCL2L11-encoded trigger enhanced apoptosis.
Conclusions:
- Discovered widespread TDMD triggers in target RNAs, expanding the understanding of miRNA regulation.
- Demonstrated a functional link between TDMD and apoptosis, where miRNA degradation can enhance programmed cell death.
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