Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule.

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogeneous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.

SLIRP, a small SRA binding protein, is a nuclear receptor corepressor.

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.

MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3'-untranslated region of the androgen receptor messenger RNA.

The androgen receptor (AR) mediates androgen action and plays a central role in the proliferation of specific cancer cells. We demonstrated recently that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells and that androgens differentially regulate AR mRNA decay dependent on cell type (Yeap, B. B., Kreuger, R. G., Leedman, P. J. (1999) Endocrinology 140, 3282-3291). Here, we have identified a highly conserved UC-rich region in the 3-untranslated region of AR mRNA that contains a 5'-C(U)(n)C motif and a 3'-CCCUCCC poly(C)-binding protein motif. In transfection studies with LNCaP human prostate cancer cells, the AR UC-rich region reduced expression of a luciferase reporter gene. The AR UC-rich region was a target for cytoplasmic and nuclear RNA-binding proteins from human prostate and breast cancer cells as well as human testicular and breast cancer tissue. One of these proteins is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C)-binding protein-1 and -2 (CP1 and CP2), previously implicated in the control of mRNA turnover and translation, also bound avidly to the UC-rich region. Mutational analysis of the UC-rich region identified specific binding motifs for both HuR and the CPs. HuR and CP1 bound simultaneously to the UC-rich RNA and in a cooperative manner. Immunoprecipitation studies confirmed that each of these proteins associated with AR mRNA in prostate cancer cells. In summary, we have identified and characterized a novel complex of AR mRNA-binding proteins that target the highly conserved UC-rich region. The binding of HuR, CP1, and CP2 to AR mRNA suggests a role for each of these proteins in the post-transcriptional regulation of AR expression in cancer cells.