"Obra Social" La Caixa, Cellex Foundation, and the Health and Science Departments of the Generalitat de Catalunya.

Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-02062-4) contains supplementary material, which is available to authorized users.

Edited by Wilhelm JustKeywords: Inosine Adenosine deaminase acting on tRNA Transfer RNA Deaminase Codon usage Evolution a b s t r a c t Inosine on transfer RNAs (tRNAs) are post-transcriptionally formed by a deamination mechanism of adenosines at positions 34, 37 and 57 of certain tRNAs. Despite its ubiquitous nature, the biological role of inosine in tRNAs remains poorly understood. Recent developments in the study of nucleotide modifications are beginning to indicate that the dynamics of such modifications are used in the control of specific genetic programs. Likewise, the essentiality of inosine-modified tRNAs in genome evolution and animal biology is becoming apparent. Here we review our current understanding on the role of inosine in tRNAs, the enzymes that catalyze the modification and the evolutionary link between such enzymes and other deaminases. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Inosine and inosine modification enzymesInosine is a non-canonical nucleoside found in all domains of life. Chemically, it is a guanosine analogue and it only differs from the latter by the lack of the N2 amino group. Inosine is rarely present in DNA but is often observed in different types of RNAs including double-stranded RNAs, tRNAs and viral RNAs [76,63,30]. In RNA, inosine is produced by the deamination of adenosine [31,5]. Generally, 2 groups of RNA adenosine deaminases exist: adenosine deaminases acting on messenger RNAs (ADARs) and adenosine deaminases acting on tRNAs (ADATs), the enzymes of each group being specific for specific modification sites [5,76,24,25].ADARs are present only in metazoans and, in vertebrates, three genes that encode for different ADAR proteins have been described. ADAR1 and ADAR2 are expressed in most tissues and their deamination activity has been confirmed. ADAR3 on the contrary is only expressed in the central nervous system and its function is currently unknown, as it lacks deamination activity [45]. As mentioned, ADARs deaminate adenosines in mRNAs to inosine. Since inosine (which is derived from adenosines), resembles guanosine, A-to-I editing on mRNAs can result in amino acid substitutions during translation, and alterations of splice sites during mRNA processing [76]. Additionally, ADARs can edit non-coding RNAs and have regulatory functions, for example, by affecting the biogenesis, processing and target selection of siRNAs and miRNAs [76].Inosine is found in tRNAs in all domains of life. It is present mainly at three positions on tRNAs: position 34, which is the first nucleotide of the anticodon (wobble-position), position 37 (following the anticodon), and position 57 (at the TWC-loop) (Fig. 1A). Interestingly, at position 34, inosine is the final modified base, while at positions 37 and 57 inosine is found in a methylated state (m 1 I37, m 1 I57 or m 1 Im57) [40,55]. Methyl-inosine 37 has only been found in eukaryotic tRNA Ala [30,40] and the modification involves two enzymatic reactions. First, A37 is deaminated to I3...

HB patients GENOMIC STUDY TRANSCRIPTOMIC STUDY METHYLATION STUDY CytoScan HD ®-array RNA-sequencing/ ddPCR HTA ®-array/ RT-qPCR 850K (EPIC)-array/ QUAlu Dysregulation of global RNA & BLCAP editing Overexpression of 14q32 DLK1-DIO3 genes 16 + VIM-gene signature (C1/C2/C2B) 2 epigenomic HB subtypes (Epi-CA & Epi-CB) CLINICAL PARAMETERS: prognostic marker identification Poor prognostic factors:-4q,-18, 17q11.2 AI (NF1) CHKA new therapeutic target Molecular risk stratification MRS1 MRS2 MRS3 Strong 14q32 Epi-CB Time Survival Highlights Hepatoblastoma (HB) involves global dysregulation of RNA editing, including in the tumor suppressor BLCAP. Overexpression of a 300 kb region within the 14q32 DLK1/DIO3 locus is a new hallmark of HB. We identified 2 epigenomic HB subtypes-Epi-CA and Epi-CB-with distinct degrees of DNA hypomethylation and CpG island hypermethylation. The molecular risk stratification of HB, based on the 14q32-signature and epigenomic subtypes, is associated with patient outcomes. The enzyme CHKA could be a novel therapeutic target for patients with HB.

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Drosophila telomere maintenance depends on the transposition of the specialized retrotransposons HeT-A, TART, and TAHRE. Controlling the activation and silencing of these elements is crucial for a precise telomere function without compromising genomic integrity. Here we describe two chromosomal proteins, JIL-1 and Z4 (also known as Putzig), which are necessary for establishing a fine-tuned regulation of the transcription of the major component of Drosophila telomeres, the HeT-A retrotransposon, thus guaranteeing genome stability. We found that mutant alleles of JIL-1 have decreased HeT-A transcription, putting forward this kinase as the first positive regulator of telomere transcription in Drosophila described to date. We describe how the decrease in HeT-A transcription in JIL-1 alleles correlates with an increase in silencing chromatin marks such as H3K9me3 and HP1a at the HeT-A promoter. Moreover, we have detected that Z4 mutant alleles show moderate telomere instability, suggesting an important role of the JIL-1-Z4 complex in establishing and maintaining an appropriate chromatin environment at Drosophila telomeres. Interestingly, we have detected a biochemical interaction between Z4 and the HeT-A Gag protein, which could explain how the Z4-JIL-1 complex is targeted to the telomeres. Accordingly, we demonstrate that a phenotype of telomere instability similar to that observed for Z4 mutant alleles is found when the gene that encodes the HeT-A Gag protein is knocked down. We propose a model to explain the observed transcriptional and stability changes in relation to other heterochromatin components characteristic of Drosophila telomeres, such as HP1a.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetal HMGA2 gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogene RPSAP52 . RPSAP52 is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation of RPSAP52 impairs the balance between the oncogene LIN28B and the tumor suppressor let-7 family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels of RPSAP52 in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.