The RNA-binding protein TDP-43 selectively disrupts microRNA-1/206 incorporation into the RNA-induced silencing complex

Abstract

MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity.

Keywords: Cardiac muscle; Gene Regulation; MicroRNA; RNA-Protein Interaction; RNA-binding Protein; Skeletal Muscle.

FIGURE 1.

TDP-43 interacts with the miR-1/miR-206 family, but not miR-133. A, sequence alignment of the miR-1/miR-206 family or the miR-133 family. miRNA seed sequence is highlighted in green. Residues that differ among family members are highlighted in yellow. B, EMSAs revealed an miRNA-protein complex (arrow) in undifferentiated C₂C₁₂ cell lysate that interacted with fluorescently labeled miR-1 or miR-206 probe (Fluo-miR-1, Fluo-miR-206). These interactions could be competed with unlabeled miR-1 or miR-206, but not with unlabeled miR-133a. 5′-biotinylated miR-1 (Bio-miR-1) also effectively competed for binding. C, eluates from negative control (Biotin), Bio-miR-133a, or Bio-miR-1 pulldowns were run on denaturing gels. Proteins enriched in the Bio-miR-1 pulldown lane were identified by mass spectrometry and included KSRP and TDP-43. D, Western analysis detecting TDP-43 protein following cross-linking and immunoprecipitation from C₂C₁₂ skeletal myoblasts using α-TDP-43 (TDP-43 IP), rabbit IgG (IgG IP), or 10% input showed efficient and specific recovery of TDP-43 protein. E, qRT-PCR to detect miR-206 and miR-133b after TDP-43 cross-linking and immunoprecipitation performed in D revealed preferential interaction of TDP-43 with miR-206 versus miR-133b. Results were normalized to pulldown with IgG (NS, not significant; *, p < 0.05).

FIGURE 2.

Loss of TBPH, the Drosophila Tdp-43 ortholog, increases miR-1 activity in fly wings. A, wings from control or dppGAL4::UAS-dmiR-1-expressing flies (dpp>dmiR-1) show that miR-1 expression causes decreased L3-L4 intervein distance (25). B, fly wings from intercrosses of three different TBPH hypomorphic lines to dpp>dmiR-1 flies showed enhancement of the dpp>dmiR-1 phenotype. Quantitation of wing phenotypes from parental, dpp>dmiR-1 (Control), or dpp>dmiR-1 X TBPH hypomorph crosses is shown. C, wing morphology in flies carrying the temperature-sensitive dpp>dmiR-1 allele housed at 18, 20, or 22 °C. Quantitation of wing phenotypes is shown. Narrow is defined as L3-L4 intervein distance of dpp>dmiR-1 at 18 °C; Enhanced Narrowing is defined as ≥50% further reduction in L3-L4 intervein distance as compared with dpp>dmiR-1 at 18 °C.

FIGURE 3.

TDP-43 suppresses activity of miR-1 and miR-206 by inhibiting their association with AGO2. A, Western blots to detect TDP-43 and AGO2 in C₂C₁₂ cells transfected with nontargeting control siRNA (siControl) or siRNA directed against Tdp-43 (siTDP-43). Arrow indicates 43-kDa band corresponding to TDP-43. GAPDH levels show equal protein loading. B, miR-206 and miR-133b levels in C₂C₁₂ cells transfected with nontargeting control siRNA (siControl) or siRNA directed against Tdp-43 (siTDP-43) as detected by qRT-PCR and normalized to U6 levels. C, luciferase reporter assays measuring activity of miR-1 or miR-206 in control (siControl) or TDP-43-depleted (siTDP-43) cells. Ctrl miRNA, control miRNA. D, Western analysis detecting AGO2 protein following immunoprecipitation from TDP-43-depleted (siTDP-43) or control (siControl) C₂C₁₂ cells using α-AGO2 (AGO2 IP), mouse IgG (IgG IP), or 20% input showed efficient and specific recovery of AGO2 protein. E, miRNA levels recovered by AGO2 RNA IP from C₂C₁₂ cells transfected with an siRNA targeting Tdp-43 (siTDP-43) were compared with those transfected with a nontargeting control siRNA (siControl) (NS, not significant; *, p < 0.05).

FIGURE 4.

TDP-43 overexpression decreases miR-1 family activity in mouse muscle. A, average levels of IGF-1 protein, determined by ELISA, and Igf-1 mRNA, detected by qRT-PCR, in hindlimb skeletal muscle of 3.5 week old, male, nontransgenic (NTG, n = 2) or TDP-43 transgenic (TDP-43 TG, n = 3) littermates. B, Western blots to detect HDAC4 and TDP-43 protein in hindlimb skeletal muscle of 3.5 week old, male, nontransgenic (NTG) or TDP-43 transgenic (TDP-43 TG) littermates. Relative HDAC4 quantity, as determined by densitometry, is shown above. GAPDH levels show equal protein loading. C, average levels of HDAC4 protein and Hdac4 mRNA in samples analyzed in A and B. D, relative levels of miR-1 and miR-206 detected by qRT-PCR using the same samples analyzed in A–C (NS, not significant; *, p < 0.05).