Project description:During the early stages of embryonic development, Lin28a is expressed at high levels and gradually decreases as the embryo develops. As an RNA-binding protein, Lin28a maintains a subset of adult muscle stem cells (MuSCs) in an embryonic-like state. However, the specific mechanism for regulating RNA metabolism is not yet clear. Through the analysis of Lin28a-associated genes, we have revealed that Lin28a promotes the expression of Igf2bp3 and interacts with the Igf2bp3 protein, controlling the proliferation of MuSCs. In response to stress stimuli, Lin28a rapidly upregulates the expression of Igf2bp3, recruits mRNAs by interacting with the N6-methyladenosine (m6A) reader Igf2bp3, and forms protein complexes with G3bp1 in stress granules. Sequencing of the transcriptome and RNAs immunoprecipitated by Lin28a, Igf2bp3, and m6A antibodies in Lin28a+ MuSCs further revealed that Lin28a and Igf2bp3 collaboratively regulate the expression of DNA repair-related genes such as Fancm and Usp1 to promote DNA repair after oxidative stress. Therefore, Lin28a regulates the expression of DNA damage repair-related genes and upregulates the DNA stress response of MuSCs through stress granule regulation of m6A-modified mRNAs. This positive regulation contributes to the self-renewal of MuSCs.
Project description:Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of the mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal the enrichment of Lin28A binding around transcription start sites, and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and provide a new framework for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems. Examine the DNA binding ability of Lin28 and its roles in regulating gene expression by coordinating with Tet1
Project description:Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of the mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal the enrichment of Lin28A binding around transcription start sites, and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and provide a new framework for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems.
Project description:To identify the directly bound transcripts of Flag antibody (the backgroud control for our METTL16 RIP-seq), RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T. Briefly, HEK293T cells were infected with pmiRNA1-empty vector. Only the GFP-positive cells were used for study and expanded in DMEM medium.