Disrupting miR-466l-3p and HuR Cooperation with Target Site Blockers Reveals a Therapeutic Strategy to Destabilize mRNA Transcripts

MicroRNAs (miRNAs) typically regulate gene expression by promoting mRNA degradation, but select miRNAs, such as miR-466l-3p (miR-466), can instead stabilize transcripts in coordination with RNA-binding proteins (RBPs) like HuR. We identify conserved AU-rich elements (cAREs) within the 3'UTRs of IL-17A, GM-CSF, and IL-23A as critical cis-regulatory binding sites where miR-466 facilitates HuR recruitment to promote mRNA stability. Using site-directed mutagenesis, RNA pulldown, and MS2-TRAP assays to capture miRNA-mRNA complexes, we demonstrate that HuR binding depends on prior engagement by miR-466. Disrupting this interaction with rationally designed Target Site Blockers (TSBs) oligonucleotides destabilizes target mRNAs and suppresses cytokine expression in vitro and in vivo. TSBs directed against IL-17A, GM-CSF, and IL-23A selectively blocked miR-466 binding, reduced transcript stability, and lowered cytokine production without affecting unrelated mRNAs. In murine models of LPS-induced inflammation, psoriasis, and autoimmunity, TSBs exhibited therapeutic efficacy and cytokine specificity, outperforming monoclonal antibodies in some settings. Phosphorothioate-modified TSBs enabled systemic delivery and retained activity in human T cells, underscoring translational potential. Similar to antisense oligonucleotides, TSBs trigger RNase H1-mediated degradation while also blocking miRNA-mRNA interactions. These findings establish miR-466-HuR cooperation as a therapeutically targetable axis through TSBs without affecting global miRNA function. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/709388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@82c326org.highwire.dtl.DTLVardef@da15e7org.highwire.dtl.DTLVardef@1d3fc69org.highwire.dtl.DTLVardef@607656_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TEXTBOXMechanism of TSB-mediated disruption of cooperative miRNA-HuR-dependent mRNA stabilizationA: In the canonical model, destabilizing miRNAs (e.g., miR-16) bind to their target sites within the 3'UTR, recruiting the RNA-induced silencing complex (miRISC) to promote mRNA decay or translational repression. B: In contrast, a newly identified class of miRNAs--stabilizing miRNAs (E-miRNAs), such as miR-466l-3p--bind to specific target sequences within AU-rich elements (AREs) in the 3'UTR. This binding facilitates cooperative recruitment of the RNA-binding protein HuR (ELAVL1), resulting in enhanced mRNA stability and/or translation. C: Target site blockers (TSBs) designed to occlude miRNA-binding sites competitively inhibit miRISC loading, thereby disrupting HuR engagement and reversing stabilization. This selective disruption leads to transcript-specific mRNA destabilization without affecting global miRNA function. C_TEXTBOX