TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes

Abstract

Although aberrant microRNA (miRNA) expression is linked to human diseases including cancer, the mechanisms that regulate the expression of each individual miRNA remain largely unknown. TAR DNA-binding protein-43 (TDP-43) is homologous to the heterogeneous nuclear ribonucleoproteins (hnRNPs), which are involved in RNA processing, and its abnormal cellular distribution is a key feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two neurodegenerative diseases. Here, we show that TDP-43 facilitates the production of a subset of precursor miRNAs (pre-miRNAs) by both interacting with the nuclear Drosha complex and binding directly to the relevant primary miRNAs (pri-miRNAs). Furthermore, cytoplasmic TDP-43, which interacts with the Dicer complex, promotes the processing of some of these pre-miRNAs via binding to their terminal loops. Finally, we show that involvement of TDP-43 in miRNA biogenesis is indispensable for neuronal outgrowth. These results support a previously uncharacterized role for TDP-43 in posttranscriptional regulation of miRNA expression in both the nucleus and the cytoplasm.

Fig. 1.

Association between TDP-43 and the Drosha complex. (A and B) Nuclear extracts (NE) from HEK293T-derived stable cell lines that expressed FLAG-Drosha (A) or FLAG-TDP-43 (B) and from nontransfected HEK293T cells were subjected to immunoprecipitation (IP) with anti-FLAG antibody followed by immunoblot (IB) analysis. Samples that corresponded to 5% of the NE used in the assay were loaded as Input. (A) FLAG-Drosha, whose expression was approximately fivefold higher than that of endogenous Drosha, was represented by two forms (band a and band b) as previously described (5). (B) Band a, full-length FLAG-TDP-43; band b, endogenous TDP-43; band c, cleaved fragment of FLAG-TDP-43. (C) Complexes that contained FLAG-Drosha were subjected to Superose 6 size-exclusion chromatography and analyzed by immunoblotting (IB). The fractions from the column are indicated at the bottom. (D) Immunoprecipitates of nuclear complexes that contained FLAG-Drosha were digested with the indicated amount of either RNase V1 or RNase A (0.1–10 units), separated into supernatant (Sup.) and bead (Beads) fractions, and subjected to IB analysis. (E) Schematic diagram of wild-type TDP-43 tagged with Myc at the N terminus and the regions deleted in each construct. The regions that were indispensable for the association with the Drosha complex (amino acids 316–402) and the Dicer complex (amino acids 261–402) are indicated in red. Gly-rich, glycine-rich domain; A2B, hnRNP A2-binding domain; M9-like, M9-like domain; NES, nuclear export signal; NLS, nuclear localization signal; RRM, RNA recognition motif (32). (F) A wide range of deletion mutants of Myc-TDP-43, which are indicated at the top, were transfected into HEK293T-derived stable cell lines that expressed FLAG-Drosha. Immunoprecipitates of nuclear complexes that contained FLAG-Drosha were subjected to IB analysis.

Fig. 2.

Identification of TDP-43–regulated miRNAs. (A) The expression levels of the mature forms of the indicated miRNAs in SH-SY5Y cells transfected with either TDP-43 siRNA or control siRNA are shown (n = 5). (B) An RIP assay was performed in SH-SY5Y cells whereby RNA fragments were immunoprecipitated with anti–TDP-43 antibody or control IgG and subsequently subjected to quantitative RT-PCR analysis for the indicated pri-miRNAs (n = 3). (C) Nuclear extracts (NE) and cytoplasmic extracts (CE) from HEK293T-derived stable cell lines that expressed FLAG-TDP-43 (T) and from nontransfected HEK293T cells (C) were subjected to immunoprecipitation (IP) with anti-FLAG antibody, after which the RNA fragments were purified. RT-PCR amplification was performed using RT primers specific for the pri-miRNAs (lanes 2–7) or that recognized both pri-miRNAs and pre-miRNAs (lanes 8–13). EMSA was performed with the indicated amounts of recTDP-43 and radiolabeled wild-type (WT) pre-miR-574 (D and E) or mutant pre-miR-574 RNAs (MUT1 and MUT2) (E). The percentage amounts of RNA that bound to the indicated amounts of recTDP-43 protein were then quantified (n = 3). (D) (Upper) The sequence of human pre-miR-574 is shown. The regions known to be processed into mature miR-574-5p and miR-574-3p are shown in green. (E) (Upper) The expected hairpin structures of MUT1 and MUT2 are shown, and the mutated sites are highlighted in red.

Fig. 3.

Association between TDP-43 and the Dicer complex. (A and B) Cytoplasmic extracts (CE) from HEK293T-derived stable cell lines that expressed FLAG-Dicer (A) or FLAG-TDP-43 (B) and from nontransfected HEK293T cells were subjected to immunoprecipitation (IP) with anti-FLAG antibody followed by immunoblot (IB) analysis. Samples that corresponded to 5% of the CE used in the assay were loaded as Input. (A) The expression of FLAG-Dicer was approximately fourfold higher than that of endogenous Dicer. (B) Band a, full-length FLAG-TDP-43; band b, endogenous TDP-43; band c, cleaved fragment of FLAG-TDP-43. (C) Complexes that contained FLAG-Dicer were subjected to Superose 6 size-exclusion chromatography and analyzed by immunoblotting (IB). The fractions from the column are indicated at the bottom. (D) Immunoprecipitates of cytoplasmic complexes that contained FLAG-Dicer were digested with the indicated amounts of either RNase V1 or RNase A (0.1–10 units), separated into supernatant (Sup.) and bead (Beads) fractions, and subjected to IB analysis. (E) A wide range of deletion mutants of TDP-43 were transfected into HEK293T-derived stable cell lines that expressed FLAG-Dicer. Immunoprecipitates of cytoplasmic complexes that contained FLAG-Dicer were subjected to IB analysis. The deleted regions in each Myc-TDP-43 mutant are indicated at the top.

Fig. 4.

Analysis of the miRNA processing activity with or without TDP-43. HEK293T-derived stable cell lines that expressed either FLAG-Drosha or FLAG-Dicer were transfected with TDP-43 siRNA1 or control siRNA. (A and C) Subsequently, nuclear extracts (NE) (A) or cytoplasmic extracts (CE) (C) were subjected to immunoprecipitation (IP) with anti-FLAG antibody followed by immunoblot (IB) analysis. (B) The relative cleavage efficiency for Drosha complex that lacked TDP-43 against Drosha complex that contained TDP-43 is plotted for each pri-miRNA (n = 3). (D) The relative cleavage efficiency for the Dicer complex that lacked TDP-43 against the Dicer complex that contained TDP-43 is plotted for each pre-miRNA (n = 3). (E) Each siRNA-resistant Myc-tagged TDP-43 construct shown at the top was cotransfected with TDP-43 siRNA1 into SH-SY5Y cells. The expression of the Myc-tagged TDP-43 proteins and the knockdown of endogenous TDP-43 were confirmed by IB analysis. (F) Total RNA was extracted from SH-SY5Y cells transfected with TDP-43 siRNA1 and each siRNA-resistant Myc-tagged TDP-43 construct and was subjected to quantitative RT-PCR analysis for the indicated miRNAs (n = 5).

Fig. 5.

Requirement of TDP-43–regulated miRNAs for neuronal differentiation. (A) Images of Neuro2a cells 48 h after differentiation that was induced 24 h after transfection with either TDP-43 siRNA or control siRNA. (B) The average length of the longest neurites 48 h after differentiation is plotted (n = 101). (C and D) Differential interference-contrast (DIC) images of Neuro2a cells (Left) 48 h after differentiation that was induced 24 h after cotransfection of TDP-43 siRNA with either pmR-ZsGreen1-pri-miR-132 (C) or pmR-ZsGreen1-pri-miR-143 (D). Plasmid transfection was ensured by ZsGreen1 expression (Right). (E) The average length of the longest neurites 48 h after differentiation is plotted (n = 101). (Scale bars, 20 μm.)