Menin is required for optimal processing of the microRNA let-7a

Abstract

Multiple endocrine neoplasia type I (MEN1) is an inherited syndrome that includes susceptibility to pancreatic islet hyperplasia. This syndrome results from mutations in the MEN1 gene, which encodes menin protein. Menin interacts with several transcription factors, including JunD, and inhibits their activities. However, the precise mechanism by which menin suppresses gene expression is not well understood. Here, we show that menin interacts with arsenite-resistant protein 2 (ARS2), a component of the nuclear RNA CAP-binding complex that is crucial for biogenesis of certain miRNAs including let-7a. The levels of primary-let-7a (pri-let-7a) are not affected by menin; however, the levels of mature let-7a are substantially decreased upon Men1 excision. Let-7a targets, including Insr and Irs2, pro-proliferative genes that are crucial for insulin-mediated signaling, are up-regulated in Men1-excised cells. Inhibition of let-7a using anti-miRNA in wild type cells is sufficient to enhance the expression of insulin receptor substrate 2 (IRS2) to levels observed in Men1-excised cells. Depletion of menin does not affect the expression of Drosha and CBP80, but substantially impairs the processing of pri-miRNA to pre-miRNA. Ars2 knockdown decreased let-7a processing in menin-expressing cells but had little impact on let-7a levels in menin-excised cells. As IRS2 is known to mediate insulin signaling and insulin/mitogen-induced cell proliferation, these findings collectively unravel a novel mechanism whereby menin suppresses cell proliferation, at least partly by promoting the processing of certain miRNAs, including let-7a, leading to suppression of Irs2 expression and insulin signaling.

Keywords: Beta Cell; Cancer Biology; IRS2; Menin; MicroRNA; Pancreas; Pancreatic Islets; Tumor Suppressor Gene; let-7a.

FIGURE 1.

Menin interacts with ARS2. A, silver staining of menin-containing fractions after affinity purification using anti-FLAG M2 beads in HEK293 cells ectopically expressing menin. Visible bands were excised for identification by mass spectroscopy. Asterisk indicates ARS2 peptide fragments identified by mass spectroscopy; MLDAAVIK, LTPLLSVR, NINGITQHK, RGWVTFDR, VALSEPQPER, SKYHPDEVGK, FVTSNTQELGK, VLDKLLLYLR, DLDAPDDVDFF, VRNINGITQHK, VALSEPQPERR, ISHGEVLEWQK, ESLSEEEAQKMGR, EVAFFNNFLTDAK, TFEEKLTPLLSVR, LGSIAEIDLGVPPPVMK, MLDAAVIKMEGGTENDLR, AIVEYRDLDAPDDVDFF, ILEQEEEEEQAGKPGEPSK, and ILEQEEEEEQAGKPGEPSKK. B, nuclear extract from HEK293 cells ectopically expressing menin were immunoprecipitated (IP) with anti-menin antibody, and immunoblotted for ARS2. Asterisk indicates a nonspecific band.

FIGURE 2.

Mature let-7a miRNA levels are increased in menin-expressing cells. A, Men1^l/l and Men1^l/l;CreER MEFs were treated with 4-hydroxytamoxifen (4-OHT), and excision of menin was determined by Western blotting. B and C, qRT-PCR showing the levels of let-7a (B) and miR-155 (C) in Men1^l/l and Men1^l/l;CreER MEFs treated with 4-OHT. D, qRT-PCR showing let-7a levels in BON cells ectopically expressing either vector or wild type (WT) menin; inset, Western blotting showing levels of ectopic menin expression. Error bars indicate ± S.D.

FIGURE 3.

Menin does not affect the levels of the pri-miRNA transcript. Men1^l/l and Men1^l/l;CreER MEFs were treated with 4-hydroxytamoxifen, and the levels of pri-let7a (A) and pri-miR-155 (B) were determined by qRT-PCR. Error bars indicate ± S.D.

FIGURE 4.

Menin is required for processing of let-7a. Radiolabeled pri-let-7a was incubated with cell extracts from WT and menin-null MEFs, and the RNA was resolved on a 6% urea-polyacrylamide gel followed by autoradiography. The substrate, pri-let-7a, and product, pre-let-7a, are as indicated. nt, nucleotides.

FIGURE 5.

Men1 excision does not affect the levels of pri-miRNA processing machinery. Men1^l/l and Men1^l/l;CreER MEFs were treated with 4-hydroxytamoxifen, and the protein levels of Drosha and nuclear cap-binding protein subunit 1 (CBP80) were analyzed by immunoblotting. Actin was used as a loading control.

FIGURE 6.

Ars2 knockdown results in decreased let-7a processing in menin-expressing cells. A, immunoblotting for ARS2 in menin-expressing (Men1^l/l) and menin-null cells (Men1^Δ/Δ) expressing either control or Ars2-targeting shRNAs. Immunoblotting for actin is included as a loading control. B, qRT-PCR showing the levels of let-7a in Men1^l/l and Men1^Δ/Δ cells expressing either control or Ars2-targeting shRNAs.

FIGURE 7.

IRS2 levels are reduced in menin-expressing cells. A, Men1^l/l and Men1^l/l;CreER MEFs were treated with 4-hydroxytamoxifen, and the mRNA levels of Irs2 were determined by qRT-PCR. B, immunoblotting for IRS2 in cell lysates from Men1^l/l and Men1^l/l;CreER MEFs is shown. Ponceau S is included as a loading control. C, retrovirus expressing either control or menin was stably transduced into βHC9, and the mRNA levels of Irs2 were determined by qRT-PCR; inset, Western blotting showing protein levels of IRS2. D, cell lysates from BON cells ectopically expressing either vector or menin were immunoblotted for menin and IRS2. Ponceau S staining is included as a loading control. Error bars indicate ± S.D.

FIGURE 8.

Anti-miR-induced repression of let-7a expression results in increased levels of IRS2. Men1^l/l and Men1^Δ/Δ MEFs were harvested 5 days after transfection with either control (−) or let-7a (+) anti-miR, and the protein levels of IRS2 and RAS were determined by Western blotting. Immunoblotting for actin is included as a loading control.