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Background: RNA pseudouridylation is a critical post‐transcriptional modification that influences gene expression and impacts various biological functions. Despite its significance, the role of mRNA pseudouridylation in cancer remains poorly understood. This study investigates the impact of pseudouridine synthase 7 (PUS7)‐mediated pseudouridylation of Alpha‐ketoglutarate‐dependent Dioxygenase alkB Homolog 3 (ALKBH3) mRNA in gastric cancer. Methods: Immunohistochemistry and Western blotting were used to assess PUS7 protein levels in human gastric cancer tissues. The relationship between PUS7 and gastric cancer progression was examined using 3D colony formation assays and subcutaneous xenograft models. Real‐time quantitative PCR (RT‐qPCR), Western blotting, and polysome profiling assays were conducted to investigate how PUS7 regulates ALKBH3. A locus‐specific pseudouridine (Ψ) detection assay was used to identify Ψ sites on ALKBH3 mRNA. Results: Our findings indicate a significant reduction of PUS7 in gastric cancer tissues compared to adjacent non‐tumour tissues. Functional analyses reveal that PUS7 inhibits gastric cancer cell proliferation and tumour growth via its catalytic activity. Additionally, PUS7 enhances the translation efficiency of ALKBH3 mRNA by modifying the U696 site with pseudouridine, thereby attenuating tumour growth. Importantly, ALKBH3 functions as a tumour suppressor in gastric cancer, with its expression closely correlated with PUS7 levels in tumour tissues. Conclusions: PUS7‐dependent pseudouridylation of ALKBH3 mRNA enhances its translation, thereby suppressing gastric cancer progression. These findings highlight the potential significance of mRNA pseudouridylation in cancer biology and suggest a therapeutic target for gastric cancer. Highlights: PUS7 enhances the translation efficiency of ALKBH3 through its pseudouridylation activity on ALKBH3 mRNA, thereby inhibiting gastric tumourigenesis.The expression levels of PUS7 and ALKBH3 are significantly correlated in gastric tumours, which may be potential prognostic predictors and therapeutic targets for patients with gastric cancer. [ABSTRACT FROM AUTHOR]

Primary cilia are antenna-like subcellular structures to act as signaling platforms to regulate many cellular processes and embryonic development. m1A RNA modification plays key roles in RNA metabolism and gene expression; however, the physiological function of m1A modification remains largely unknown. Here we find that the m1A demethylase ALKBH3 significantly inhibits ciliogenesis in mammalian cells by its demethylation activity. Mechanistically, ALKBH3 removes m1A sites on mRNA of Aurora A, a master suppressor of ciliogenesis. Depletion of ALKBH3 enhances Aurora A mRNA decay and inhibits its translation. Moreover, alkbh3 morphants exhibit ciliary defects, including curved body, pericardial edema, abnormal otoliths, and dilation in pronephric ducts in zebrafish embryos, which are significantly rescued by wild-type alkbh3, but not by its catalytically inactive mutant. The ciliary defects caused by ALKBH3 depletion in both vertebrate cells and embryos are also significantly reversed by ectopic expression of Aurora A mRNA. Together, our data indicate that ALKBH3-dependent m1A demethylation has a crucial role in the regulation of Aurora A mRNA, which is essential for ciliogenesis and cilia-associated developmental events in vertebrates. [ABSTRACT FROM AUTHOR]

N1‐methyladenosine (m1A) is a prevalent and reversible RNA modification, which plays a crucial role in the regulation of RNA fate and gene expression. However, the lack of tools to precisely manipulate m1A sites in specific transcripts has hindered efforts to clarify the association between a specific m1A‐modified transcript and its phenotypic outcomes. Here we develop a CRISPR‐Cas13d‐based tool called reengineered m1A modification valid eraser (termed "REMOVER") for targeted m1A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m1A demethylase ALKBH3, and the dCasRx‐ALKBH3 fusion protein can mediate potent demethylation of m1A‐modified RNAs. We further find that REMOVER can specifically demethylate m1A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m1A‐modified transcripts, which enables further elucidation of the relationship between m1A modification of specific transcripts and their phenotypic outcomes. [ABSTRACT FROM AUTHOR]

N 1 -methyladenosine (m 1 A) is a prevalent and reversible RNA modification, which plays a crucial role in the regulation of RNA fate and gene expression. However, the lack of tools to precisely manipulate m 1 A sites in specific transcripts has hindered efforts to clarify the association between a specific m 1 A-modified transcript and its phenotypic outcomes. Here we develop a CRISPR-Cas13d-based tool called reengineered m 1 A modification valid eraser (termed "REMOVER") for targeted m 1 A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m 1 A demethylase ALKBH3, and the dCasRx-ALKBH3 fusion protein can mediate potent demethylation of m 1 A-modified RNAs. We further find that REMOVER can specifically demethylate m 1 A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m 1 A-modified transcripts, which enables further elucidation of the relationship between m 1 A modification of specific transcripts and their phenotypic outcomes., (© 2021 Wiley-VCH GmbH.)