ABSTRACT
Although differentiation of pluripotent embryonic stem cells is restricted by a hierarchy of transcription factors, little is known about whether post-transcriptional mechanisms similarly regulate early embryoid differentiation. We developed a system where small hairpin (sh)RNAs can be induced in embryonic stem (ES) cells from a defined locus following integration by Flp recombinase-mediated DNA recombination. To verify the system, the key transcription factor Stat3, which maintains pluripotency, was downregulated by shRNA, and the expected morphological and biochemical markers of differentiation were observed. Induction of shRNA specific for the post-transcriptional regulator Brf1 (Zfp36L1) amplified the cardiac markers with strong stimulation of cardiomyocyte formation within embryoid bodies. These findings identify Brf1 as a novel potential regulator of cardiomyocyte formation and suggest that post-transcriptional mechanisms are of importance to early development and, possibly, to regenerative medicine. The inducible RNA interference system presented here should also allow assignment of function for candidate genes with suspected roles in ES cell development. Disclosure of potential conflicts of interest is found at the end of this article.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Genetic Techniques , RNA Interference , Animals , Butyrate Response Factor 1 , Cell Differentiation/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Doxycycline/pharmacology , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryonic Stem Cells/drug effects , Leukemia Inhibitory Factor/metabolism , Mice , Models, Biological , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , STAT3 Transcription Factor/geneticsABSTRACT
Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3' untranslated regions that act as mRNA stability determinants by interacting with ARE binding proteins (ARE-BPs). The mechanisms underlying the function of ARE and ARE-BP interactions in promoting mRNA decay are not fully understood. Here, we demonstrate that KSRP, a KH domain-containing ARE-BP, is an essential factor for ARE-directed mRNA decay. Some of the KH motifs (KHs) of KSRP directly mediate RNA binding, mRNA decay, and interactions with the exosome and poly(A) ribonuclease (PARN). The ability of KHs to promote mRNA decay correlates with their ability to bind the ARE and associate with RNA-degrading enzymes. Thus, KHs promote rapid mRNA decay by recruiting degradation machinery to ARE-containing mRNAs.
