YTHDF 1‐mediated translation amplifies Wnt‐driven intestinal stemness

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Abstract

N6‐methyladenosine (m 6 A) mRNA methylation has emerged as an important player in many biological processes by regulating gene expression. However, its roles in intestinal stem cell ( ISC ) homeostasis remain largely unknown. Here, we report that YTHDF 1, an m 6 A reader, is highly expressed in ISC s and its expression is upregulated by Wnt signaling at the translational level. Whereas YTHDF 1 is dispensable for normal intestinal development in mice, genetic ablation of Ythdf1 dramatically blocks Wnt‐driven regeneration and tumorigenesis with reduced ISC stemness. Mechanistically, YTHDF 1 facilitates the translation of Wnt signaling effectors including TCF 7L2/ TCF 4 , while this process is enhanced during Wnt activation to augment β‐catenin activity. Targeting YTHDF 1 in ISC s of established tumors leads to tumor shrinkage and prolonged survival. Collectively, our studies unveil YTHDF 1 as an amplifier of Wnt/β‐catenin signaling at the translational level, which is required for the maintenance of ISC s during regeneration and tumorigenesis.

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Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.

J Bokar, M Shambaugh, D Polayes … (1997)

The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to the yeast protein SPO8, which is involved in induction of sporulation by an unknown mechanism.