Project description:N6-methyladenosine (m6A) is the major type of RNA modi'cation that regulates RNA stability and gene expression, characterized by a conserved motif including 5'-(m6A)C-3'. However, the functional significance of the specific motif for m6A modification is unclear. Here, we focused on a single amino acid substitution in the m6A 'writer' complex, METTL14 R298P, and created mutation knock-in cells. m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) revealed that METTL14R298P/R298P but not METTL14WT/R298P cells had an aberrant sequence motif, 5'-(m6A)[C/T]-3'. This study reveals a mechanism underlying sequence-specific m6A modification.
Project description:N6-methyladenosine (m6A) is the major type of RNA modification that regulates RNA stability and gene expression, characterized by a conserved motif including 5′-(m6A)C-3′. However, the functional significance of the specific motif for m6A modification is unclear. Here, we focused on a single amino acid substitution in the m6A “writer” complex, METTL14 R298P, and created mutation knock-in cells. m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) revealed that METTL14R298P/R298P but not METTL14WT/R298P cells had an aberrant sequence motif, 5′-(m6A)[C/T]-3′. This study reveals a mechanism underlying sequence-specific m6A modification.
Project description:β-cell specific Mettl14 knock-out mice display reduced N6-methyladenosine (m6A) levels and recapitulate human Type II diabetes (T2D) islet phenotype with early diabetes onset and mortality secondary to decreased β-cell proliferation and insulin degranulation. To gain insights into the role of m6A in regulating the IGF1/insulin -> AKT - > PDX1 pathway and to dissect the signaling networks modulating AKT phosphorylation, we subjected freshly isolated islets from control and Mettl14 knock-out mice to phospho-antibody microarrays.
Project description:Post-transcriptional m6A methylation on mRNA plays a key role in neural development. Here, we specifically depleted Mettl14 (a key component of m6A complex) in retina progenitor cells by crossing Mettl14 fl/fl mice with the Chx10-Egfp/Cre mouse line and investigated Mettl14 depletion-induced m6A alteration in developing retinas using m6A-seq.
Project description:Aberrant RNA splicing may contribute to carcinogenesis. Here, we focused on the G96V mutation in SNRPD3, one of the splicing factors. We found that RNA splicing is altered under hypoxia in cell lines in which the gene mutation is knocked-in.
Project description:Post-transcriptional m6A methylation on mRNA plays a key role in neural development. Here, we specifically depleted Mettl14 (a key component of m6A complex) in retina progenitor cells by crossing Mettl14 fl/fl mice with the Chx10-Egfp/Cre mouse line and investigated cell type and transcriptome changes in the developing retinas using scRNA-seq.