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. 2021 Dec 1;35(23-24):1595–1609. doi: 10.1101/gad.348874.121

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© 2021 Li et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — Sequence requirement for TDMD triggers. Schematic of the WT BCL2L11 trigger (A) and TRIM9 trigger (C) constructs with a mutated miR-221/222 or miR-218 site. The miR-221/222 site in BCL2L11 or the miR-218 site in TRIM9 was mutated to either disrupt or expand pairing to the corresponding miRNA or introduce an extensively complementary site to miR-16. The importance of the complementary site for the BCL2L11 trigger (B) or TRIM9 trigger (D) function. Representative Northern blot measuring miR-221, miR-222, miR-218, miR-16, miR-92a, and let-7a levels in HEK293T cells transfected with the constructs described in A and C. The normalized miRNA abundance and standard deviation are shown below each miRNA band. miRNA abundance in B and D was normalized to miR-92a and let-7a, respectively. The miRNA abundance in control samples was normalized to 1. n = 3 biological replicates. (E) Schematic of long and short TDMD trigger constructs. The long TDMD trigger includes the trigger sequence as well as the conserved flanking regions, while the short TDMD trigger contains only the TDMD trigger sequence. (F) The importance of the TDMD-flanking region on miRNA degradation. Representative Northern blot measuring miR-7, miR-218, miR-221, miR-222, and miR-16 levels in HEK293T cells transfected with either the long or short TDMD constructs described in E. The normalized miRNA abundance and standard deviation are shown below each miRNA band. miRNA abundance was normalized to miR-16. n = 3 biological replicates.