m6A RNA methylation regulates mitochondrial function.

RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses.

Protocol for detecting RBM33-binding sites in HEK293T cells using PAR-CLIP-seq.

RNA-binding proteins (RBPs) regulate gene expression both co-transcriptionally and post-transcriptionally. Here, we provide a protocol for photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by next-generation sequencing (PAR-CLIP-seq). PAR-CLIP-seq is a transcriptome-scale technique for identifying in vivo binding sites of RBPs at the single-nucleotide level.

RBM33 is a unique mA RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of mA demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized mA-binding protein that plays a critical role in ALKBH5-mediated mRNA mA demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its mA-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation.

The METTL5-TRMT112 N-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N-methyladenosine (mA), and Ndimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single mA sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA mA modification, the methyltransferase responsible for the 18S rRNA mA modification has remained unclear.

KAS-seq: genome-wide sequencing of single-stranded DNA by N-kethoxal-assisted labeling.

Transcription and its dynamics are crucial for gene expression regulation. However, very few methods can directly read out transcriptional activity with low-input material and high temporal resolution. This protocol describes KAS-seq, a robust and sensitive approach for capturing genome-wide single-stranded DNA (ssDNA) profiles using N-kethoxal-assisted labeling.

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm-Controlled Gene Expression in Mice.

N-methyladenosine (mA), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(mA)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown.

Lysine acetylation restricts mutant IDH2 activity to optimize transformation in AML cells.

Mutant isocitrate dehydrogenase (IDH) 1 and 2 play a pathogenic role in cancers, including acute myeloid leukemia (AML), by producing oncometabolite 2-hydroxyglutarate (2-HG). We recently reported that tyrosine phosphorylation activates IDH1 R132H mutant in AML cells. Here, we show that mutant IDH2 (mIDH2) R140Q commonly has K413 acetylation, which negatively regulates mIDH2 activity in human AML cells by attenuating dimerization and blocking binding of substrate (α-ketoglutarate) and cofactor (NADPH).

Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases mA Methylation.

N-methyladenosine (mA) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of mA methylation in mRNA metabolism has been well documented recently, regulation of the mA machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the mA deposition.

RNA Demethylase ALKBH5 Selectively Promotes Tumorigenesis and Cancer Stem Cell Self-Renewal in Acute Myeloid Leukemia.

N-methyladenosine (mA), the most abundant internal modification in mRNA, has been implicated in tumorigenesis. As an mA demethylase, ALKBH5 has been shown to promote the development of breast cancer and brain tumors. However, in acute myeloid leukemia (AML), ALKBH5 was reported to be frequently deleted, implying a tumor-suppressor role.

The RNA-binding protein FMRP facilitates the nuclear export of -methyladenosine-containing mRNAs.

-Methyladenosine (mA) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to mA-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans.

mA mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.

N-methyladenosine (mA) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of mA mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in mA methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex.