Project description:The total RNAs were immunoprecipitated with anti-N6-methyadenosine (m6A) antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were labeled with Cy5 and Cy3 respectively as cRNAs in separate reactions using Arraystar Super RNA Labeling Kit. The cRNAs were combined together and hybridized onto Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray (8x60K, Arraystar). After washing the slides, the arrays were scanned in two-color channels by an Agilent Scanner G2505C.
Project description:We report a previously unrecognized role of IGF2BP3 in survival of neuroblastoma cells. To explore IGF2BP3-dependent gene regulation and m6A modification, RNA-seq and m6A-seq analysis was conducted and the differently expressed/modified genes were revealed in the IGF2BP3-knockdown SK-N-BE(2) cells in comparison to control group.
Project description:Chemically modified mRNA nucleotides are emerging as key regulators of gene expression. Their effects are typically thought to be mediated through reader proteins that selectively bind to RNA molecules containing these modifications. Here, we present a new mechanism by which N6 methyladenosine (m6A) couples the translation of messenger RNA (mRNA) to its decay. m6A-modified codons are decoded inefficiently by the ribosome, rendering them “non-optimal”. At these codons, m6A slows down elongating ribosomes and thereby triggers translation-dependent decay of the mRNA. Inefficient decoding of m6A-modified codons is counteracted by the transfer RNA (tRNA) anticodon modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). mcm5s2U increases the decoding efficiency of a subset of m6A codons and thereby alleviates m6A-mediated mRNA decay. This unanticipated link between the mRNA and tRNA epitranscriptomes controls the decay of mRNAs of important oncogenic signaling pathways. The biogenesis of m6A and mcm5s2U is coordinated in normal tissues but dysregulated in cancer, where a shift towards more mcm5s2U stabilizes these RNAs and is associated with higher tumor aggressiveness and poor prognosis. Thus, the balance between m6A and mcm5s2U acts as a pan-epitranscriptomic mechanism that regulates gene expression and curtails tumorigenesis.
Project description:Both N6-methyladenosine (m6A) mediates RNA fates and ubiquitin mediates protein fates play an important role in either physiology or pathology including cancer, yet how long noncoding RNAs (lncRNAs) are involved in a link of molecular fate between m6A and ubiquitin remains unknown. Here, we reveal a role for a lncRNA Downregulated RNA in Cancer (DRAIC) to suppress tumor growth and metastasis in clear cell Renal Carcinoma (ccRCC). Mechanistically, DRAIC physically interacts with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) and enhances its protein stability by blocking E3 ligase F-box protein 11 (FBXO11)-mediated ubiquitin and proteasome-dependent degradation. Subsequently, hnRNPA2B1 destabilizes m6A modified-type 1 insulin-like growth factor receptor (IGF1R) to lead to inhibition of ccRCC progression. Moreover, four m6A modification sites of IGF1R are identified and results in its mRNA degradation. Collectively, our findings reveal that DRAIC/hnRNPA2B1 axis regulates IGF1R mRNA expression in an m6A-dependent manner and highlights an important mechanism of IGF1R fate. These findings shed light on DRAIC/hnRNPA2B1/FBXO11/IGF1R axis as potential therapeutic targets in ccRCC and build a link of molecular fate between m6A-modified RNA and ubiquitin-modified protein.
Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing. Identification of m6A modified sequences in HepG2 cells.
Project description:Roles of m6A-modified mRNA transcripts in the context of MI were preliminarily verified. In the context of m6A methylation, three hub mRNAs were validated to impact the process of apoptosis/angiogenesis. Our study provided theoretical basis and innovative targets for treatment of MI and paved the way for future investigations aiming at exploring upstream epigenetic mechanisms of pathogenesis after MI.
Project description:Chemically modified mRNA nucleotides are emerging as key regulators of gene expression. Their effects are typically thought to be mediated through reader proteins that selectively bind to RNA molecules containing these modifications. Here, we present a new mechanism by which N6 methyladenosine (m6A) couples the translation of messenger RNA (mRNA) to its decay. m6A-modified codons are decoded inefficiently by the ribosome, rendering them “non-optimal”. At these codons, m6A slows down elongating ribosomes and thereby triggers translation-dependent decay of the mRNA. Inefficient decoding of m6A-modified codons is counteracted by the transfer RNA (tRNA) anticodon modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). mcm5s2U increases the decoding efficiency of a subset of m6A codons and thereby alleviates m6A-mediated mRNA decay. This unanticipated link between the mRNA and tRNA epitranscriptomes controls the decay of mRNAs of important oncogenic signaling pathways. The biogenesis of m6A and mcm5s2U is coordinated in normal tissues but dysregulated in cancer, where a shift towards more mcm5s2U stabilizes these RNAs and is associated with higher tumor aggressiveness and poor prognosis. Thus, the balance between m6A and mcm5s2U acts as a pan-epitranscriptomic mechanism that regulates gene expression and curtails tumorigenesis.
Project description:Chemically modified mRNA nucleotides are emerging as key regulators of gene expression. Their effects are typically thought to be mediated through reader proteins that selectively bind to RNA molecules containing these modifications. Here, we present a new mechanism by which N6 methyladenosine (m6A) couples the translation of messenger RNA (mRNA) to its decay. m6A-modified codons are decoded inefficiently by the ribosome, rendering them “non-optimal”. At these codons, m6A slows down elongating ribosomes and thereby triggers translation-dependent decay of the mRNA. Inefficient decoding of m6A-modified codons is counteracted by the transfer RNA (tRNA) anticodon modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). mcm5s2U increases the decoding efficiency of a subset of m6A codons and thereby alleviates m6A-mediated mRNA decay. This unanticipated link between the mRNA and tRNA epitranscriptomes controls the decay of mRNAs of important oncogenic signaling pathways. The biogenesis of m6A and mcm5s2U is coordinated in normal tissues but dysregulated in cancer, where a shift towards more mcm5s2U stabilizes these RNAs and is associated with higher tumor aggressiveness and poor prognosis. Thus, the balance between m6A and mcm5s2U acts as a pan-epitranscriptomic mechanism that regulates gene expression and curtails tumorigenesis.
Project description:Chemically modified mRNA nucleotides are emerging as key regulators of gene expression. Their effects are typically thought to be mediated through reader proteins that selectively bind to RNA molecules containing these modifications. Here, we present a new mechanism by which N6 methyladenosine (m6A) couples the translation of messenger RNA (mRNA) to its decay. m6A-modified codons are decoded inefficiently by the ribosome, rendering them “non-optimal”. At these codons, m6A slows down elongating ribosomes and thereby triggers translation-dependent decay of the mRNA. Inefficient decoding of m6A-modified codons is counteracted by the transfer RNA (tRNA) anticodon modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). mcm5s2U increases the decoding efficiency of a subset of m6A codons and thereby alleviates m6A-mediated mRNA decay. This unanticipated link between the mRNA and tRNA epitranscriptomes controls the decay of mRNAs of important oncogenic signaling pathways. The biogenesis of m6A and mcm5s2U is coordinated in normal tissues but dysregulated in cancer, where a shift towards more mcm5s2U stabilizes these RNAs and is associated with higher tumor aggressiveness and poor prognosis. Thus, the balance between m6A and mcm5s2U acts as a pan-epitranscriptomic mechanism that regulates gene expression and curtails tumorigenesis.