Calreticulin Interacts with C/EBPalpha and C/EBPbeta MRNAs and Represses Translation of C/EBP Proteins

Molecular and Cellular Biology. Oct, 2002  |  Pubmed ID: 12242300

We previously identified an RNA binding protein, CUGBP1, which binds to GCN repeats located within the 5' region of C/EBPbeta mRNAs and regulates translation of C/EBPbeta isoforms. To further investigate the role of RNA binding proteins in the posttranscriptional control of C/EBP proteins, we purified additional RNA binding proteins that interact with GC-rich RNAs and that may regulate RNA processing. In HeLa cells, the majority of GC-rich RNA binding proteins are associated with endogenous RNA transcripts. The separation of these proteins from endogenous RNA identified several proteins in addition to CUGBP1 that specifically interact with the GC-rich 5' region of C/EBPbeta mRNA. One of these proteins was purified to homogeneity and was identified as calreticulin (CRT). CRT is a multifunctional protein involved in several biological processes, including interaction with and regulation of rubella virus RNA processing. Our data demonstrate that both CUGBP1 and CRT interact with GCU repeats within myotonin protein kinase and with GCN repeats within C/EBPalpha and C/EBPbeta mRNAs. GCN repeats within these mRNAs form stable SL structures. The interaction of CRT with SL structures of C/EBPbeta and C/EBPalpha mRNAs leads to inhibition of translation of C/EBP proteins in vitro and in vivo. Deletions or mutations abolishing the formation of SL structures within C/EBPalpha and C/EBPbeta mRNAs lead to a failure of CRT to inhibit translation of C/EBP proteins. CRT-dependent inhibition of C/EBPalpha is sufficient to block the growth-inhibitory activity of C/EBPalpha. This finding further defines the molecular mechanism for posttranscriptional regulation of the C/EBPalpha and C/EBPbeta proteins.

Myotonic Dystrophy: Discussion of Molecular Basis

Advances in Experimental Medicine and Biology. 2002  |  Pubmed ID: 12611434

Overexpression of CUG Triplet Repeat-binding Protein, CUGBP1, in Mice Inhibits Myogenesis

The Journal of Biological Chemistry. Mar, 2004  |  Pubmed ID: 14722059

Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients.

RNA CUG-binding Protein 1 Increases Translation of 20-kDa Isoform of CCAAT/enhancer-binding Protein Beta by Interacting with the Alpha and Beta Subunits of Eukaryotic Initiation Translation Factor 2

The Journal of Biological Chemistry. May, 2005  |  Pubmed ID: 15788409

Expression of a dominant negative 20-kDa isoform of CCAAT/enhancer-binding protein (C/EBPbeta), LIP, is increased in proliferating livers and in tumor cells. Two RNA-binding proteins, CUGBP1 and calreticulin, have been implicated in the translational regulation of C/EBPbeta. In this paper, we present evidence showing several critical steps by which liver increases translation of LIP after partial hepatectomy. At early stages after partial hepatectomy, liver activates CUGBP1 by a hyperphosphorylation. The activated CUGBP1 binds to the 5' region of C/EBPbeta mRNA and replaces calreticulin, which partially represses translation of C/EBPbeta in quiescent livers. The hyperphosphorylated CUGBP1 also interacts with the alpha and beta subunits of initiation factor eIF2. Our data demonstrate that the interaction of CUGBP1 with the eIF2alpha enhances the association of CUGBP1 with ribosomes and correlates with increased translation of LIP in the liver after partial hepatectomy. Our data support the hypothesis that CUGBP1 increases translation of LIP by the interaction with the eIF2alpha subunit. This facilitates subsequent recruitment of larger numbers of ribosomes to initiate translation of LIP.

Age-specific CUGBP1-eIF2 Complex Increases Translation of CCAAT/enhancer-binding Protein Beta in Old Liver

The Journal of Biological Chemistry. Oct, 2006  |  Pubmed ID: 16931514

The RNA-binding protein CUGBP1 regulates translation of proteins in a variety of biological processes. In this study, we show that aging liver increases CUGBP1 translational activities by induction of a high molecular weight protein-protein complex of CUGBP1. The complex contains CUGBP1, subunits alpha, beta, and gamma of the initiation translation factor eIF2, and four proteins of the endoplasmic reticulum, eR90, CRT, eR60, and Grp78. The induction of the CUGBP1-eIF2 complex in old livers is associated with the elevation of protein levels of CUGBP1 and with the hyper-phosphorylation of CUGBP1 by a cyclin D3-cdk4 kinase, activity of which is increased with age. We have examined the role of the elevation of CUGBP1 and the role of cyclin D3-cdk4-mediated phosphorylation of CUGBP1 in the formation of the CUGBP1-eIF2 complex by using CUGBP1 transgenic mice and young animals expressing high levels of cyclin D3 after injection with cyclin D3 plasmid. These studies showed that both the increased levels of CUGBP1 and cdk4-mediated hyper-phosphorylation of CUGBP1 are involved in the age-associated induction of the CUGBP1-eIF2 complex. The CUGBP1-eIF2 complex is bound to C/EBPbeta mRNA in the liver of old animals, and this binding correlates with the increased amounts of liver-enriched activator protein and liver-enriched inhibitory protein. Consistent with these observations, the purified CUGBP1-eIF2 complex binds to the 5' region of C/EBPbeta mRNA and significantly increases translation of the three isoforms of C/EBPbeta in a cell-free translation system, in cultured cells, and in the liver. Thus, these studies demonstrated that age-mediated induction of the CUGBP1-eIF2 complex changes translation of C/EBPbeta in old livers.

Ectopic Expression of Cyclin D3 Corrects Differentiation of DM1 Myoblasts Through Activation of RNA CUG-binding Protein, CUGBP1

Experimental Cell Research. Jul, 2008  |  Pubmed ID: 18570922

Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBPbeta mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.

Expression of RNA CCUG Repeats Dysregulates Translation and Degradation of Proteins in Myotonic Dystrophy 2 Patients

The American Journal of Pathology. Aug, 2009  |  Pubmed ID: 19590039

Myotonic dystrophy 2 (DM2) is a multisystem skeletal muscle disease caused by an expansion of tetranucleotide CCTG repeats, the transcription of which results in the accumulation of untranslated CCUG RNA. In this study, we report that CCUG repeats both bind to and misregulate the biological functions of cytoplasmic multiprotein complexes. Two CCUG-interacting complexes were subsequently purified and analyzed. A major component of one of the complexes was found to be the 20S catalytic core complex of the proteasome. The second complex was found to contain CUG triplet repeat RNA-binding protein 1 (CUGBP1) and the translation initiation factor eIF2. Consistent with the biological functions of the 20S proteasome and the CUGBP1-eIF2 complexes, the stability of short-lived proteins and the levels of the translational targets of CUGBP1 were shown to be elevated in DM2 myoblasts. We found that the overexpression of CCUG repeats in human myoblasts from unaffected patients, in C2C12 myoblasts, and in a DM2 mouse model alters protein translation and degradation, similar to the alterations observed in DM2 patients. Taken together, these findings show that RNA CCUG repeats misregulate protein turnover on both the levels of translation and proteasome-mediated protein degradation.

CCUG Repeats Reduce the Rate of Global Protein Synthesis in Myotonic Dystrophy Type 2

Reviews in the Neurosciences. 2010  |  Pubmed ID: 20458885

Expansion of non-coding CTG and CCTG repeats in the 3' UTR of the myotonin protein kinase (DMPK) gene in Myotonic Dystrophy type 1 (DM1) and in the intron 1 of Zinc Finger Protein 9 (ZNF9) in Myotonic Dystrophy type 2 (DM2) represent typical non-coding mutations that cause the diseases mainly through transdominant effect on the RNA metabolism (splicing, translation and RNA stability). The commonly recognized RNA gain-of-function mechanism of DM1 and DM2 suggests that the mutant CUG and CCUG RNAs play a critical role in myotonic dystrophies (DMs) without a significant role of DMPK and ZNF9. Recent studies have shown that the molecular pathogenesis of DM2 also involves the protein product of the ZNF9 gene. CCUG repeats reduce ZNF9 protein, a translational regulator of the terminal oligo-pyrimidine tract (TOP) mRNAs encoding proteins of translational apparatus. Thus, in DM2 cells, expansion of CCUG repeats affects not only multiple RNAs, but also down-regulates ZNF9 which in turn reduces translation of the TOP-containing mRNAs and diminishes the rate of global protein synthesis. In this review, we discuss the role of expansion of CCUG repeats in the reduction of ZNF9-mediated regulation of the rate of protein synthesis in DM2 and the consequences of this reduction in the multi-systemic phenotype of DM2.