Abstract
We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.
Highlights
MID1, a regulator of phosphatase 2A (PP2A), forms part of a messenger ribonucleoprotein complex
PDPK-1, an important regulator of mTOR/ PP2A signaling, is among the MIDAS-containing mRNAs, and we have found that PDPK-1 protein synthesis is significantly reduced in cells from an Opitz BBB/G syndrome (OS) patient, which further potentiates mTOR dysfunction in MID1 mutation carriers
These results show that the MIDAS motif can increase cellular production of proteins encoded by transfected genes, independent of the following: (i) its position within the mRNA, (ii) the particular protein produced, and (iii) the vector used for protein expression
Summary
MID1, a regulator of PP2A, forms part of a messenger ribonucleoprotein complex. Results: MID1 complex binds a purine-rich sequence motif in mRNAs and regulates their translation. Our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. Our data suggest that MID1 targets specific mRNAs to the microtubule network and regulates their efficient translation, functions that would suggest loss of cytoskeleton-bound protein translation as a novel mechanism underlying the developmental defects observed in Opitz syndrome patients [8]. PDPK-1, an important regulator of mTOR/ PP2A signaling, is among the MIDAS-containing mRNAs, and we have found that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient, which further potentiates mTOR dysfunction in MID1 mutation carriers. By targeting protein translation, inclusion of one or more MIDAS sequences into the 3ЈUTR of a gene of interest can significantly increase productivity of the respective proteins in eukaryotic cells and can be used to fine-tune protein expression in eukaryotic cell systems
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