i ⁶ A-seq maps <i>N</i> ⁶ -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

Abstract Dynamic post-transcriptional RNA modifications are crucial regulators of RNA metabolism and cell fate. Recent advances in sequencing, combined with antibody, enzymatic, or chemical approaches, have enabled transcriptome-wide mapping of these modifications. While prevalent in tRNAs and rRNAs, a growing number of modifications also adorn lower-abundance mRNA transcripts. Global mapping efforts, particularly for N 6 -methyladenosine (m 6 A), uncovered the affected mechanisms governing RNA metabolism, shedding light on novel, modification-dependent, modes of gene regulation. N 6 -isopentenyladenosine (i 6 A) is a conserved tRNA modification involved in translation fidelity and efficiency. i 6 A depletion is linked to mitochondrial defects and human diseases. Current i 6 A detection methods are low-throughput, and its lack of base-pairing effects makes sequencing-based identification challenging. Here we developed a novel and robust mapping technique, i 6 A-seq, a method that utilizes antibody-mediated enrichment and iodine chemical labeling to generate a reverse transcription signature, enabling transcriptome-wide i 6 A detection. Our global mapping revealed hundreds of i 6 A sites in human and mouse mRNA, indicating its presence in this RNA species, with conserved isopentenylome features. These sites exhibit a typical consensus sequence, primarily in coding transcripts (CDS), preferentially within lysine codons. Consistent with its role in tRNA, tRNA-isopentenyltransferase (TRIT1) also appears to install i 6 A in mRNA. Manipulation of TRIT1 revealed that i 6 A regulates the expression of a subset of genes through mRNA decay, specifically those isopentenylated at the CDS and translated on ER-bound ribosomes. Importantly, DIS3 like 3’-5’ exoribonuclease 2 (DIS3L2), an RNA exoribonuclease known to regulate ER-translated mRNA, was identified as the first i 6 A reader protein. Our findings introduce i 6 A as a new modified nucleotide that decorates mRNA, allowing us to decipher its regulatory roles in gene expression. This study not only establishes the presence and location of i 6 A in mRNA, but also uncovers its first functional mechanism. Similar to the knowledge accumulated on m 6 A, this work paves the way for further discoveries with potential relevance for understanding gene regulation, disease diagnosis, and therapy.