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Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA.

ABSTRACT: N(6)A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N(6)-methylated adenosine reader domain and report its solution structure in complex with a N(6)-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m(6)A recognition. These findings establish a molecular function for YTH domains as m(6)A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m(6)A recognition.

SUBMITTER: Theler D

PROVIDER: S-EPMC4267619 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA.

Theler Dominik D Dominguez Cyril C Blatter Markus M Boudet Julien J Allain Frédéric H-T FH

Nucleic acids research 20141111 22

N(6)A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most pote ...[more]

PMID: 25389274

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Project description:BackgroundA number of studies have demonstrated that N6-methyladenosine (m6A) plays a vital role in the pathological process of various tumours. Recently, it was found that m6A writers or erasers affect the tumourigenesis of melanoma. However, the relationship between m6A readers such as YTH domain family (YTHDF) proteins and melanoma was still elusive.MethodsRT-qPCR, Western blot and immunohistochemistry were conducted to measure the expression level of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) and lysyl oxidase-like 3 (LOXL3) in melanoma tissues and cells. The effects of YTHDF3 and LOXL3 on melanoma were verified in vitro and in vivo. Multi-omics analysis including RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses was performed to identify the target. The interaction between YTHDF3 and LOXL3 was verified by RT-PCR, Western blot, MeRIP-qPCR, RIP-qPCR and CRISPR-Cas13b-based epitranscriptome engineering.ResultsIn this study, we found that m6A reader YTHDF3 could affect the metastasis of melanoma both in vitro and in vivo. The downstream targets of YTHDF3, such as LOXL3, phosphodiesterase 3A (PDE3A) and chromodomain helicase DNA-binding protein 7 (CHD7) were identified by means of RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses. Besides, RT-qPCR, Western blot, RIP-qPCR and MeRIP-qPCR were performed for subsequent validation. Among various targets of YTHDF3, LOXL3 was found to be the optimal target of YTHDF3. With the application of CRISPR-Cas13b-based epitranscriptome engineering, we further confirmed that the transcript of LOXL3 was captured and regulated by YTHDF3 via m6A binding sites. YTHDF3 augmented the protein expression of LOXL3 without affecting its mRNA level via the enrichment of eukaryotic translation initiation factor 3 subunit A (eIF3A) on the transcript of LOXL3. LOXL3 downregulation inhibited the metastatic ability of melanoma cells, and overexpression of LOXL3 ameliorated the inhibition of melanoma metastasis caused by YTHDF3 downregulation.ConclusionsThe YTHDF3-LOXL3 axis could serve as a promising target to be interfered with to inhibit the metastasis of melanoma.

Dataset Information

Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA.

Publications

Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA.

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