RBM33 is a unique m(6)A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

Yu F ，Zhu AC ，Liu S ，Gao B ，Wang Y ，Khudaverdyan N ，Yu C ，Wu Q ，Jiang Y ，Song J ，Jin L ，He C ，Qian Z ... -  《-》

The m6A demethylase ALKBH5 promotes tumor progression by inhibiting RIG-I expression and interferon alpha production through the IKKε/TBK1/IRF3 pathway in head and neck squamous cell carcinoma.

N6-methyladenosine (m6A) RNA modification plays a critical role in various physiological and pathological conditions. However, the role of m6A modification in head and neck squamous cell carcinoma (HNSCC) remains elusive. In this study, the expression of m6A demethylases was detected by HNSCC tissue microarray. m6A-RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing were used to identify downstream targets of ALKBH5. Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) was used to explore the m6A "readers". Tumor-infiltrating lymphocytes were analyzed in SCC7-bearing xenografts in C3H mice. Here, we demonstrate the downregulation of m6A status and upregulation of two demethylases in HNSCC. Silencing the m6A demethylase alkB homolog 5, RNA demethylase (ALKBH5) suppresses tumor progression in vitro and in vivo. m6A-RNA immunoprecipitation sequencing reveals that ALKBH5 downregulates the m6A modification of DDX58 mRNA. Moreover, RIG-I, encoded by the DDX58 mRNA, reverses the protumorigenic characteristics of ALKBH5. ChIRP-MS demonstrates that HNRNPC binds to the m6A sites of DDX58 mRNA to promote its maturation. ALKBH5 overexpression inhibits RIG-I-mediated IFNα secretion through the IKKε/TBK1/IRF3 pathway. The number of tumor-infiltrating lymphocytes in C3H immunocompetent mice is reduced by ALKBH5 overexpression and restored by IFNα administration. Upregulation of AKLBH5 negatively correlates with RIG-I and IFNα expression in HNSCC patients. These findings unveil a novel mechanism of immune microenvironment regulation mediated by m6A modification through the ALKBH5/RIG-I/IFNα axis, providing a rationale for therapeutically targeting epitranscriptomic modulators in HNSCC.

Jin S ，Li M ，Chang H ，Wang R ，Zhang Z ，Zhang J ，He Y ，Ma H ... -  《Molecular Cancer》

RBM33: A new regulator of N-6-methyladenosine (m(6)A) methylation.

In this issue of Molecular Cell, Yu et al.1 identify RBM33 as a previously unrecognized m6A (N-6-methyladenosine) RNA binding protein that plays a critical role in ALKBH5-mediated m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5.

Zhang P ，Abdel-Wahab O 《-》

RNA demethylase ALKBH5 regulates hypopharyngeal squamous cell carcinoma ferroptosis by posttranscriptionally activating NFE2L2/NRF2 in an m(6) A-IGF2BP2-dependent manner.

Having emerged as the most abundant posttranscriptional internal mRNA modification in eukaryotes, N6-methyladenosine (m6 A) has attracted tremendous scientific interest in recent years. However, the functional importance of the m6 A methylation machinery in ferroptosis regulation in hypopharyngeal squamous cell carcinoma (HPSCC) remains unclear. We herein performed bioinformatic analysis, cell biological analyses, transcriptome-wide m6 A sequencing (m6 A-seq, MeRIP-seq), RNA sequencing (RNA-seq), and RNA immunoprecipitation sequencing (RIP-seq), followed by m6 A dot blot, MeRIP-qPCR, RIP-qPCR, and dual-luciferase reporter assays. The results revealed that ALKBH5-mediated m6 A demethylation led to the posttranscriptional inhibition of NFE2L2/NRF2, which is crucial for the regulation of antioxidant molecules in cells, at two m6 A residues in the 3'-UTR. Knocking down ALKBH5 subsequently increased the expression of NFE2L2/NRF2 and increased the resistance of HPSCC cells to ferroptosis. In addition, m6 A-mediated NFE2L2/NRF2 stabilization was dependent on the m6 A reader IGF2BP2. We suggest that ALKBH5 dysregulates NFE2L2/NRF2 expression in HPSCC through an m6 A-IGF2BP2-dependent mechanism. Together, these results have revealed an association between the ALKBH5-NFE2L2/NRF2 axis and ferroptosis, providing insight into the functional importance of reversible mRNA m6 A methylation and its modulators in HPSCC.

Ye J ，Chen X ，Jiang X ，Dong Z ，Hu S ，Xiao M ... -  《-》

K235 acetylation couples with PSPC1 to regulate the m(6)A demethylation activity of ALKBH5 and tumorigenesis.

N6-methyladenosine (m6A) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two m6A demethylases. Here, we reveal that ALKBH5 is acetylated at lysine 235 (K235) by lysine acetyltransferase 8 and deacetylated by histone deacetylase 7. K235 acetylation strengthens the m6A demethylation activity of ALKBH5 by increasing its recognition of m6A on mRNA. RNA-binding protein paraspeckle component 1 (PSCP1) is a regulatory subunit of ALKBH5 and preferentially interacts with K235-acetylated ALKBH5 to recruit and facilitate the recognition of m6A mRNA by ALKBH5, thereby promoting m6A erasure. Mitogenic signals promote ALKBH5 K235 acetylation. K235 acetylation of ALKBH5 is upregulated in cancers and promotes tumorigenesis. Thus, our findings reveal that the m6A demethylation activity of ALKBH5 is orchestrated by its K235 acetylation and regulatory subunit PSPC1 and that K235 acetylation is necessary for the m6A demethylase activity and oncogenic roles of ALKBH5.

Zhang XL ，Chen XH ，Xu B ，Chen M ，Zhu S ，Meng N ，Wang JZ ，Zhu H ，Chen D ，Liu JB ，Yan GR ... -  《Nature Communications》