i6A-seq maps N6-isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

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 N6-methyladenosine (m6A), uncovered the affected mechanisms governing RNA metabolism, shedding light on novel, modification-dependent, modes of gene regulation. N6-isopentenyladenosine (i6A) is a conserved tRNA modification involved in translation fidelity and efficiency. i6A depletion is linked to mitochondrial defects and human diseases. Current i6A 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, i6A-seq, a method that utilizes antibody-mediated enrichment and iodine chemical labeling to generate a reverse transcription signature, enabling transcriptome-wide i6A detection. Our global mapping revealed hundreds of i6A 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 i6A in mRNA. Manipulation of TRIT1 revealed that i6A 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 i6A reader protein. Our findings introduce i6A 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 i6A in mRNA, but also uncovers its first functional mechanism. Similar to the knowledge accumulated on m6A, this work paves the way for further discoveries with potential relevance for understanding gene regulation, disease diagnosis, and therapy.