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Title: RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels      
dateReleased:
12-22-2014
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 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 × g for 10 min at 4°C. 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 4°C. 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 37°C to remove the DNA. The samples were then incubated for 15 min at 55°C 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.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-58259
refinement:
raw
alternateIdentifiers:
58259
keywords:
functional genomics
dateModified:
01-02-2015
availability:
available
types:
gene expression
name:
Mus musculus
ID:
A-MEXP-1174
name:
Illumina MouseRef-8 v2.0 Expression BeadChip
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-58259/E-GEOD-58259.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-58259/E-GEOD-58259.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58259
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress

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