• BMB Reports
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• v.50 no.4
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• pp.158-159
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• 2017

MicroRNAs (miRNAs) regulate gene expression by guiding the Argonaute (Ago)-containing RNA-induced silencing complex (RISC) to specific target mRNA molecules. It is well established that miRNAs are stabilized by Ago proteins, but the molecular features that trigger miRNA destabilization from Ago proteins remain largely unknown. To explore the molecular mechanisms of how targets affect the stability of miRNAs in human Ago (hAgo) proteins, we employed an in vitro system that consisted of a minimal hAgo2-RISC in HEK293T cell lysates. Surprisingly, we found that miRNAs are drastically destabilized by binding to seedless, non-canonical targets. We showed that miRNAs are destabilized at their 3' ends during this process, which is largely attributed to the conformational flexibility of the L1-PAZ domain. Based on these results, we propose that non-canonical targets may play an important regulatory role in controlling the stability of miRNAs, instead of being regulated by miRNAs.

• Molecules and Cells
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• v.39 no.5
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• pp.375-381
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• 2016

MicroRNAs (miRNAs) are small non-coding RNAs (~22 nucleotides) regulating gene expression at the post-transcriptional level. By directing the RNA-induced silencing complex (RISC) to bind specific target mRNAs, miRNA can repress target genes and affect various biological phenotypes. Functional miRNA target recognition is known to majorly attribute specificity to consecutive pairing with seed region (position 2-8) of miRNA. Recent advances in a transcriptome-wide method of mapping miRNA binding sites (Ago HITS-CLIP) elucidated that a large portion of miRNA-target interactions in vivo are mediated not only through the canonical "seed sites" but also via non-canonical sites (~15-80%), setting the stage to expand and determine their properties. Here we focus on recent findings from transcriptome-wide non-canonical miRNA-target interactions, specifically regarding "nucleation bulges" and "seed-like motifs". We also discuss insights from Ago HITS-CLIP data alongside structural and biochemical studies, which highlight putative mechanisms of miRNA target recognition, and the biological significance of these non-canonical sites mediating marginal repression.

• Molecules and Cells
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• v.39 no.4
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• pp.316-321
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• 2016

The receptor activator of nuclear factor ${\kappa}B$ (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function.

• BMB Reports
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• v.53 no.12
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• pp.628-633
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• 2020

WNT11 is a member of the non-canonical Wnt family and plays a crucial role in tumor progression. However, the regulatory mechanisms underlying WNT11 expression are unclear. Tumor necrosis factor-alpha (TNFα) is a major inflammatory cytokine produced in the tumor microenvironment and contributes to processes associated with tumor progression, such as tumor invasion and metastasis. By using site-directed mutagenesis and introducing a serial deletion in the 5'-regulatory region of WNT11, we observed that TNFα activates the early growth response 1 (EGR1)-binding sequence (EBS) in the proximal region of WNT11 and that the transcription factor EGR1 is necessary for the TNFα-induced transcription of WNT11. EGR1 bound directly to the EBSs within the proximal 5'-regulatory region of WNT11 and ectopic expression of EGR1 stimulated WNT11 promoter activity, whereas the knockdown of EGR1 expression by RNA interference reduced TNFα-induced WNT11 expression in T47D breast cancer cells. We also observed that mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase mediated TNFα-induced transcription of WNT11 via EGR1. Our results suggest that EGR1 directly targets WNT11 in response to TNFα stimulation in breast cancer cells.