Programmable RNA N¹‐Methyladenosine Demethylation by a Cas13d‐Directed Demethylase

Abstract

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.

ALKBH3-dependent m1A demethylation of Aurora A mRNA inhibits ciliogenesis

“…Aurora A has been demonstrated as a key protein to promote cilia disassembly 24,25 . In the initiation of ciliary disassembly, Aurora A is phosphorylated and activated at the basal body, and then phosphorylates and activates HDAC6 (histone deacetylase 6), which in turn deacetylates and destabilizes axonemal tubulin to further facilitate ciliary disassembly 21 .…”

“…Since the demethylation activity of ALKBH3 on mRNA is inhibited by the mutation of D193A 7,24 , we generated a catalytically inactive mutant of ALKBH3 (ALKBH3-D193A) to examine whether the regulation of ALKBH3 in ciliogenesis is depend on its demethylation activity. The LC-MS/MS quantification of m 1 A mRNA modification in RPE-1 cells confirmed that the ALKBH3-D193A construct we made was indeed catalytically inactive (Supplementary Fig.…”

“…5b). The amino acid residue D193 is critical for the demethylation activity of ALKBH3 on mRNA in homo sapiens 7,24 , while the residue D185 of Alkbh3 in Danio rerio was predicted to be evolutionarily conserved with D193. Alignment analysis showed that zebrafish Alkbh3 shared high homology with human ALKBH3 (identity, 56.7%; similarity, 98%) (Fig.…”

Post-Transcriptional Modifications of RNA as Regulators of Apoptosis in Glioblastoma

“…Moreover, catalytically inactive Cas13 (dCas13) has strong and specific RNA-binding activity. This allowed the creation of fusion proteins (e.g., dRfxCas13d-ALKBH3 [ 45 ], dPspCas13b-ADAR2 [ 151 ], dRfxCas13d-ALKBH5 and dRfxCas13d-METTL3 [ 152 ]) for precise control of epitranscriptome modifications of individual target genes. Further development and research involving CRISPR/Cas13 is a promising direction for basic research on the role of individual ncRNAs, as well as a tool for targeted modification of the aberrant epitranscriptome profile of glioblastoma cells.…”

“…Writers of m1A contain tRNA Methyltransferase 6 Non-Catalytic Subunit (TRMT6)—which can be located in the nucleus or cytoplasm and which methylates adenosine in tRNA as well as in mRNA [ 39 , 40 ]—and nucleomethylin, which has only nucleolar localization and methylates 28S rRNA [ 41 , 42 ]. Erasers of m1A include ALKBH1, ALKBH3 and FTO [ 43 , 44 , 45 ]. Readers of m1A comprise YTHDF1-3 and YTHDC1 [ 46 , 47 ].…”