The alteration of RNA modification patterns is emerging as a common feature of human malignancies. If these changes affect key RNA molecules for mRNA translation, such as transfer RNA, they can have important consequences for cell transformation. TRIT1 is the enzyme responsible for the hypermodification of adenosine 37 in the anticodon region of human tRNAs containing serine and selenocysteine. Herein, we show that TRIT1 undergoes gene amplification-associated overexpression in cancer cell lines and primary samples of small-cell lung cancer. From growth and functional standpoints, the induced depletion of TRIT1 expression in amplified cells reduces their tumorigenic potential and downregulates the selenoprotein transcripts. We observed that TRIT1-amplified cells are sensitive to arsenic trioxide, a compound that regulates selenoproteins, whereas reduction of TRIT1 levels confers loss of sensitivity to the drug. Overall, our results indicate a role for TRIT1 as a small-cell lung cancer-relevant gene that, when undergoing gene amplification-associated activation, can be targeted with the differentiation agent arsenic trioxide.

High-throughput genomic technologies are increasingly used in personalized cancer medicine. However, computational tools to maximize the use of scarce tissues combining distinct molecular layers are needed. Here we present a refined strategy, based on the R-package ‘conumee’, to better predict somatic copy number alterations (SCNA) from deoxyribonucleic acid (DNA) methylation arrays. Our approach, termed hereafter as ‘conumee-K_CN’, improves SCNA prediction by incorporating tumor purity and dynamic thresholding. We trained our algorithm using paired DNA methylation and SNP Array 6.0 data from The Cancer Genome Atlas samples and confirmed its performance in cancer cell lines. Most importantly, the application of our approach in cancers of unknown primary identified amplified potentially actionable targets that were experimentally validated by Fluorescence in situ hybridization and immunostaining, reaching 100% specificity and 93.3% sensitivity.

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.