The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex

Abstract

Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1α (EF-1α) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1α. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-007-0456-6) contains supplementary material, which is available to authorized users.

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

“…S1). The MID1 protein complex binds mRNAs through G-rich RNA motifs forming stable secondary structures 16,17,26 . Thus, CAG repeat stretches, which are G-rich structures and form stable hairpins, could be recognized by the MID1-complex.…”

“…One of such mRNP complexes contains the MID1 protein, the catalytic subunit of protein phosphatase 2A (PP2Ac) and 40S ribosomal S6 kinase (S6K), a target of mammalian target of rapamycin (mTOR) kinase and PP2A 16,17 . MID1 is a ubiquitin ligase that binds the a4 protein, a regulatory subunit of PP2A.…”

“…Besides regulating PP2A and mTOR activities, the MID1-complex contains polyribosomes and several translation factors and is able to bind mRNA thereby forming a microtubuleassociated mRNP complex 17 .…”

Control of mTORC1 signaling by the Opitz syndrome protein MID1

“…The RING domain has been well characterized in ubiquitin-mediated protein degradation, whereas the other domains mediate proteinprotein interactions (3)(4)(5)(6). Opitz syndrome-derived mutations in MID1 have been identified throughout the protein and show several functional consequences such as compromised association with microtubules and/or transport along microtubules (4,(7)(8)(9). In addition to its microtubule-binding function, MID1 also functions as an E3 ligase that targets the microtubule-associated pool of the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitin-mediated degradation through an interaction with the protein α4 (3).…”

Hypospadias associated with hypertelorism, the mildest phenotype of Opitz syndrome

“…15,17 In addition, MID1 is involved in the formation of multiprotein complex serving as anchor points and associated with microtubuli. 18,19 Given the molecular knowledge about the MID1 protein, a disruption in either the N-or the C-terminus of the protein would have some influences on its interaction with microtubules in the cytoplasm, which is hypothesized to be the underlying mechanism for the midline defects including hypospadias. 20 The aim of this study was to perform direct sequencing of the MID1 gene in hypospadias cases to elucidate the role of this gene in the pathogenesis of isolated hypospadias.…”