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1.
Biochem Biophys Res Commun ; 417(1): 427-32, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22166206

ABSTRACT

Post-transcriptional regulation of gene expression by RNA-binding proteins has pivotal roles in many biological processes. We have shown that Stau1, a conserved RNA-binding protein, negatively regulates myogenesis in C2C12 myoblasts. However, its target mRNAs in regulation of myogenesis remain unknown. Here we describe that Stau1 positively regulates expression of Dvl2 gene encoding a central mediator of Wnt pathway in undifferentiated C2C12 myoblasts. Stau1 binds to 3' untranslated region (UTR) of Dvl2 mRNA and Stau1 knockdown shortened a half-life of the mRNA containing Dvl2 3' UTR. After induction of myogenic differentiation, association of Stau1 with 3' UTR of Dvl2 mRNA was decreased. Correlated with the decrease in the association, the Dvl2 mRNA level was reduced during myogenesis. A forced expression of Dvl2 markedly inhibited progression of myogenic differentiation. Our results suggest that Dvl2 has an inhibitory role in myogenesis and Stau1 coordinates myogenesis through the regulation of Dvl2 mRNA.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cell Differentiation/genetics , Gene Expression Regulation, Developmental , Muscle Development/genetics , Myoblasts/cytology , Phosphoproteins/genetics , RNA Stability , RNA-Binding Proteins/metabolism , 3' Untranslated Regions/genetics , Animals , Cell Line , Dishevelled Proteins , Gene Knockdown Techniques , Mice , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/genetics
2.
Genes Cells ; 14(2): 113-28, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19170760

ABSTRACT

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a conserved RNA-binding protein that is involved in multiple processes of gene expression, including chromatin remodeling, transcription, RNA splicing, mRNA stability and translation, together with diverse groups of molecular partners. Here we identified a previously uncharacterized protein RNA binding motif protein 42 (RBM42) as hnRNP K-binding protein. RBM42 directly bound to hnRNP K in vivo and in vitro. RBM42 also directly bound to the 3' untranslated region of p21 mRNA, one of the target mRNAs for hnRNP K. RBM42 predominantly localized within the nucleus and co-localized with hnRNP K there. When cells were treated with agents, puromycin, sorbitol or arsenite, which induced the formation of stress granules (SGs), cytoplasmic aggregates of stalled translational pre-initiation complexes, both hnRNP K and RBM42 localized at SGs. Depletion of hnRNP K by RNA interference decreased cellular ATP level following release from stress conditions. Simultaneous depletion of RBM42 with hnRNP K enhanced the effect of the hnRNP K depletion. Our results indicate that hnRNP K and RBM42 are components of SGs and suggest that hnRNP K and RBM42 have a role in the maintenance of cellular ATP level in the stress conditions possibly through protecting their target mRNAs.


Subject(s)
Adenosine Triphosphate/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Stress, Physiological/physiology , 3' Untranslated Regions/metabolism , Animals , Cells, Cultured , Gene Expression , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein K/physiology , Humans , Male , Mice , NIH 3T3 Cells , Protein Binding , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA Stability/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/physiology , Tissue Distribution
3.
Genes Cells ; 13(6): 583-92, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18422603

ABSTRACT

Sequential expression of myogenic regulatory factors (MRFs) such as MyoD and myogenin drives myogenic differentiation. Besides transcriptional activation of MRFs, this process is also coordinated by post-transcriptional regulation; MyoD and myogenin mRNAs are stabilized by RNA-binding protein HuR. Stau1 is known to regulate mRNA stability in a complex with Upf1, which is termed Stau1-mediated mRNA decay (SMD). We describe here that Stau1 is involved in the regulation of myogenesis. We found that knockdown of Stau1 promotes myogenesis including the expression of a muscle-specific marker protein, myoglobin, in C2C12 myoblasts. MyoD induces myogenin expression in response to induction of myogenesis, which is a key step to start myogenesis. The level of MyoD protein was not affected when Stau1 was depleted by siRNA, whereas the levels of myogenin mRNA and protein were increased in Stau1-knockdown cells. Interestingly, myogenin promoter activity was also increased in Stau1-knockdown cells in the absence of induction of myogenesis. Furthermore, Stau1-knockdown cells spontaneously progressed myogenesis including the expression of muscle-specific protein. Although Stau1 is involved in mRNA decay together with Upf1, Upf1-knockdown did not affect progression of myogenesis. Our results suggest that Stau1 negatively regulates myogenesis in C2C12 myoblasts through a mechanism that is different from SMD.


Subject(s)
Cell Differentiation , Muscle Development , RNA-Binding Proteins/metabolism , Animals , Cell Line , Mice , Muscle Fibers, Skeletal/metabolism , Promoter Regions, Genetic , RNA-Binding Proteins/genetics , Trans-Activators/metabolism
4.
J Biol Chem ; 282(33): 24075-82, 2007 Aug 17.
Article in English | MEDLINE | ID: mdl-17576771

ABSTRACT

Adipocyte differentiation is regulated by a complex array of extracellular signals, intracellular mediators and transcription factors. Here we describe suppression of adipocyte differentiation by TRBs, mammalian orthologs of Drosophila Tribbles. Whereas all the three TRBs were expressed in 3T3-L1 preadipocytes, TRB2 and TRB3, but not TRB1, were immediately down-regulated by differentiation stimuli. Forced expression of TRB2 and TRB3 inhibited adipocyte differentiation at an early stage. Akt activation is a key event in adipogenesis and was severely inhibited by TRB3 in 3T3-L1 cells. However, the inhibition by TRB2 was mild compared with severe inhibition by TRB3, though TRB2 suppressed adipogenesis as strongly as TRB3. Interestingly, TRB2 but not TRB3 reduced the level of C/EBPbeta, a transcription factor required for an early stage of adipogenesis, through a proteasome-dependent mechanism. Furthermore, knockdown of endogenous TRB2 by siRNA allowed 3T3-L1 cells to differentiate without full differentiation stimuli. These results suggest that inhibition of Akt activation in combination with degradation of C/EBPbeta is the basis for the strong inhibitory effect of TRB2 on adipogenesis.


Subject(s)
Adipocytes/cytology , Adipogenesis , CCAAT-Enhancer-Binding Protein-beta/antagonists & inhibitors , Cell Differentiation , Intracellular Signaling Peptides and Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , 3T3-L1 Cells , Animals , Cell Cycle Proteins/physiology , Mice , RNA, Small Interfering/pharmacology
5.
J Biol Chem ; 281(49): 37913-20, 2006 Dec 08.
Article in English | MEDLINE | ID: mdl-17028188

ABSTRACT

Adipocytes play a key role in energy homeostasis and several cytokines have been shown to regulate adipogenesis. While the interleukin (IL)-6 family of cytokines was previously reported to be involved in adipogenesis, roles of this family in adipogenesis and their mechanisms of action are not fully understood. Here we show that among the IL-6 family, oncostatin M (OSM) most strongly inhibits adipogenesis of 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). We also demonstrate that OSM inhibits adipogenesis through the Ras/extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) 5 signaling pathways. In addition, OSM inhibits the early phase of the differentiation without affecting cell proliferation throughout adipogenesis including mitotic clonal expansion. CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPbeta, and peroxisome proliferator-activated receptor (PPAR) gamma are known to be required for adipogenesis. Expression of C/EBPalpha and PPARgamma was almost completely abrogated by OSM. In contrast, neither the mRNA nor protein level of C/EBPbeta was affected by OSM. Forced expression of C/EBPbeta induced differentiation in the presence of troglitazone, and OSM inhibited this C/EBPbeta-induced differentiation. Taken together, our results indicate that OSM inhibits the onset of terminal differentiation of adipocytes through the Ras/ERK and STAT5 signaling pathways by possibly regulating C/EBPbeta activity.


Subject(s)
Adipogenesis/drug effects , Adipogenesis/physiology , Oncostatin M/pharmacology , Signal Transduction/drug effects , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Adipogenesis/genetics , Animals , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cells, Cultured , MAP Kinase Signaling System/drug effects , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , STAT5 Transcription Factor/metabolism , ras Proteins/metabolism
6.
Mol Cell Biol ; 24(8): 3277-85, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15060150

ABSTRACT

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , Genes, cdc , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Cell Cycle/physiology , Checkpoint Kinase 2 , DNA Repair , DNA, Single-Stranded/metabolism , Intracellular Signaling Peptides and Proteins , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
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