Project description:The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D, hnRNP D) binds to numerous mRNAs and influences their post-transcriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RIP (RNP immunoprecipitation) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor Mef2c. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3’UTR of Mef2c mRNA and promoted Mef2c translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring Mef2c expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that Mef2c is a key effector of the myogenesis program promoted by AUF1. Keywords: ribonucleoprotein complex; post-transcriptional gene regulation; muscle cell differentiation; myocytes; mRNA translation; mRNA stability; post-transcriptional gene regulation; transcriptome
Project description:The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D, hnRNP D) binds to numerous mRNAs and influences their post-transcriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RIP (RNP immunoprecipitation) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor Mef2c. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3M-bM-^@M-^YUTR of Mef2c mRNA and promoted Mef2c translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring Mef2c expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that Mef2c is a key effector of the myogenesis program promoted by AUF1. Keywords: ribonucleoprotein complex; post-transcriptional gene regulation; muscle cell differentiation; myocytes; mRNA translation; mRNA stability; post-transcriptional gene regulation; transcriptome C2C12 mouse myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% serum (Invitrogen) and antibiotics (Invitrogen). Differentiation was induced on sub-confluent cultures by replacing the growth media (GM, DMEM with 10% FBS) with differentiation media (DM, DMEM with 2% horse serum). At various time points after differentiation, cells were harvested and RNA was extracted with phenol-chloroform and either saved as input samples for microarrys or subjected to AUF1 or IgG ribonucleoprotein immunoprecipitation. C2C12 cells cultured in GM and DM were lysed in 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40 for 10 min on ice and centrifuged at 15,000 M-CM-^W g for 10 min at 4M-BM-0C. The supernatants were incubated with protein-A Dynabeads beads coated with anti-AUF1 (Millipore) or with control IgG (Santa Cruz Biotechnology) antibodies for 2 hr at 4M-BM-0C. The beads were washed with NT2 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), followed by incubation with 20 units of RNase-free DNase I for 15 min at 37M-BM-0C to remove the DNA. The samples were then incubated for 15 min at 55M-BM-0C with 0.1% SDS/0.5 mg/ml Proteinase K to digest proteins. The RNA from the IP samples was extracted using phenol-chloroform, precipitated, and used along with the RNA from the input samples for cDNA microarrays.
Project description:RNA was isolated from material that had been immunoprecipitated (IP) from Hela cells using antibodies recognizing RNA-binding proteins HuR or AUF1, as well as using a control IgG1 antibody. RNA was reverse-transcribed in the presence of [alpha-33P]dCTP and the radiolabeled product used to hybridize human cDNA arrays. The experiment was repeated using three independent sample sets. The samples were numbered HuR-1, HuR-2, HuR-3, AUF1-1, AUF1-2, AUF1-3, IgG1-1, IgG1-2, IgG1-3. HuR represents RNA from IP reactions using an anti-HuR antibody, AUF1 represents RNA from IP reactions using an anti-AUF1 antibody and, IgG1 represents RNA from IP reactions using an anti-IgG1 antibody. The numbers 1, 2 and 3 correspond to the three independent experimental datasets. Keywords = RNa-binding protein Keywords = mRNA stability Keywords = exosome Keywords = polysome Keywords = RNA motif Keywords: ordered
Project description:RNA was isolated from material that had been immunoprecipitated (IP) from Hela cells using antibodies recognizing RNA-binding proteins HuR or AUF1, as well as using a control IgG1 antibody. RNA was reverse-transcribed in the presence of [alpha-33P]dCTP and the radiolabeled product used to hybridize human cDNA arrays. The experiment was repeated using three independent sample sets. The samples were numbered HuR-1, HuR-2, HuR-3, AUF1-1, AUF1-2, AUF1-3, IgG1-1, IgG1-2, IgG1-3. HuR represents RNA from IP reactions using an anti-HuR antibody, AUF1 represents RNA from IP reactions using an anti-AUF1 antibody and, IgG1 represents RNA from IP reactions using an anti-IgG1 antibody. The numbers 1, 2 and 3 correspond to the three independent experimental datasets. Keywords = RNa-binding protein Keywords = mRNA stability Keywords = exosome Keywords = polysome Keywords = RNA motif Keywords: ordered
Project description:Upon muscle injury the high mobility group box 1 (HMGB1) protein is up-regulated and secreted to initiate reparative responses. Here we show that HMGB1 controls myogenesis both in vitro and in vivo, during development and after adult muscle injury. HMGB1 expression in muscle cells is regulated at the translational level: the miRNA miR-1192 inhibits HMGB1 translation and the RNA-binding protein HuR promotes it. HuR binds to a cis-element, HuRBS, located in the 3'UTR of the HMGB1 transcript, and at the same time miR-1192 is recruited to an adjacent seed element. The binding of HuR to the HuRBS prevents the recruitment of Argonaute 2 (Ago2), overriding miR-1192-mediated translation inhibition. Depleting HuR reduces myoblast fusion and silencing miR-1192 re-establishes the fusion potential of HuR-depleted cells. We propose that HuR promotes the commitment of myoblasts to myogenesis by enhancing the translation of HMGB1 and suppressing the translation inhibition mediated by miR-1192. RNA content was extracted following immunoprecipitation of HuR using a monoclonal antibody (3A2) and the levels of mRNA were compared to an IgG control in order to determine which transcripts were enriched in the HuR ribonucleoprotein complex.
Project description:Upon muscle injury the high mobility group box 1 (HMGB1) protein is up-regulated and secreted to initiate reparative responses. Here we show that HMGB1 controls myogenesis both in vitro and in vivo, during development and after adult muscle injury. HMGB1 expression in muscle cells is regulated at the translational level: the miRNA miR-1192 inhibits HMGB1 translation and the RNA-binding protein HuR promotes it. HuR binds to a cis-element, HuRBS, located in the 3'UTR of the HMGB1 transcript, and at the same time miR-1192 is recruited to an adjacent seed element. The binding of HuR to the HuRBS prevents the recruitment of Argonaute 2 (Ago2), overriding miR-1192-mediated translation inhibition. Depleting HuR reduces myoblast fusion and silencing miR-1192 re-establishes the fusion potential of HuR-depleted cells. We propose that HuR promotes the commitment of myoblasts to myogenesis by enhancing the translation of HMGB1 and suppressing the translation inhibition mediated by miR-1192. RNA content was extracted following immunoprecipitation of HuR using a monoclonal antibody (3A2) and the levels of mRNA were compared to an IgG control in order to determine which transcripts were enriched in the HuR ribonucleoprotein complex.
Project description:Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. Total RNA samples were collected from primary human muscle cells (myoblasts and myotubes). Each sample has three biological replicates.
Project description:CVB 3C protease (3Cpro) plays a specific cleavage role on AU-rich binding factor (AUF1, also called as hnRNP D), consequently disputes the regulations of AUF1 on downstream molecules stabilities and transcriptions. In our work, iTRAQ approach was firstly used to quantitatively identify differentially expressed cellular proteins in AUF1-overexpressed HeLa cells, which would provide us a new insight into the role of AUF1 on cellular progress. We identified 1290 differentially expressed proteins (DEPs), including 882 upregulated and 408 downregulated DEPs at 24 h post infection (p.i.), The DEPs are involved in a variety of cellular functions such as metabolic processes, transcription, and RNA binding and exhibit different subcellular localization via GO term, protein interaction network, and series analysis. Among DEPs, DDX5, POLR2, and PPP2CA showed important roles in cellular metabolism. Especially, DDX5 was negatively regulated by AUF1 and increased during CVB infection, which in turns played an inhibitory effect on CVB replication. These findings are particularly important in the development new anti-viral therapy strategies targeting on series of new molecules.
Project description:Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. ChIP-Seq profiling of MyoD in human myotube, myoblast and rhabdomyosarcoma cells
Project description:Following skeletal muscle injury, muscle stem cells (satellite cells) are activated, proliferate, and differentiate to form myofibers. We show that mRNA decay protein AUF1 regulates satellite cell function through targeted degradation of specific mRNAs. AUF1 targets certain mRNAs containing 3 AU-rich elements (AREs) for rapid decay. Auf1-/- (KO) mice undergo accelerated skeletal muscle wasting with age and impaired muscle repair following injury. Satellite cell mRNA analysis and regeneration studies demonstrate that auf1-/- satellite cell self-renewal is impaired due to increased stability and overexpression of ARE-mRNAs. Control of ARE-mRNA decay by AUF1 and potentially other ARE-binding proteins represents a mechanism for adult stem cell regulation and is implicated in human muscle wasting diseases. We report the RNA transcript expression profiles from sorted satellite cells isolated from wild type (WT) and AUF1-null (KO) mice hindlimb muscles