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N6-methyladenosine (m 6 A), the most abundant modification of mRNA, is essential for normal development and dysregulation promotes cancer. m 6 A is highly enriched in the 3’ untranslated region (UTR) of a large subset of mRNAs to influence mRNA stability and/or translation. However, the mechanism responsible for the observed m 6 A distribution remains enigmatic. Here we find the exon junction complex shapes the m 6 A landscape by blocking METTL3-mediated m 6 A modification close to exon junctions within coding sequence (CDS). Depletion of EIF4A3, a core component of the EJC, causes increased METTL3 binding and m 6 A modification of short internal exons, and sites close to exon-exon junctions within mRNA. Reporter gene experiments further support the role of splicing and EIF4A3 deposition in controlling m 6 A modification via the local steric blockade of METTL3. Our results explain how characteristic patterns of m 6 A mRNA modification are established and uncover a role of the EJC in shaping the m 6 A epitranscriptome. Here the authors show the exon junction complex (EJC) component, EIF4A3, locally restricts METTL3- mediated mRNA methylation at exon junctions to explain the observed widespread enrichment of m6A modification in 3’ untranslated regions.

The most prevalent post-transcriptional mRNA modification, N 6 -methyladenosine (m 6 A), plays diverse RNA-regulatory roles, but its genetic control in human tissues remains uncharted. Here we report 129 transcriptome-wide m 6 A profiles, covering 91 individuals and 4 tissues (brain, lung, muscle and heart) from GTEx/eGTEx. We integrate these with interindividual genetic and expression variation, revealing 8,843 tissue-specific and 469 tissue-shared m 6 A quantitative trait loci (QTLs), which are modestly enriched in, but mostly orthogonal to, expression QTLs. We integrate m 6 A QTLs with disease genetics, identifying 184 GWAS-colocalized m 6 A QTL, including brain m 6 A QTLs underlying neuroticism, depression, schizophrenia and anxiety; lung m 6 A QTLs underlying expiratory flow and asthma; and muscle/heart m 6 A QTLs underlying coronary artery disease. Last, we predict novel m 6 A regulators that show preferential binding in m 6 A QTLs, protein interactions with known m 6 A regulators and expression correlation with the m 6 A levels of their targets. Our results provide important insights and resources for understanding both cis and trans regulation of epitranscriptomic modifications, their interindividual variation and their roles in human disease. Analysis of 129 N 6 -methyladenosine (m 6 A) profiles across 4 tissues (brain, lung, muscle and heart) identifies 8,843 tissue-specific and 469 tissue-shared m 6 A quantitative trait loci (QTLs). Of these, 184 m 6 A QTLs colocalize with GWAS signals.

MicroRNAs (miRNAs) play critical roles in development, and dysregulation of miRNA expression has been observed in human malignancies. Recent evidence suggests that the processing of several primary miRNA transcripts (pri-miRNAs) is blocked posttranscriptionally in embryonic stem cells, embryonal carcinoma cells, and primary tumors. Here we show that Lin28, a developmentally regulated RNA binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we found that Lin28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin28 as a negative regulator of miRNA biogenesis and suggest that Lin28 may play a central role in blocking miRNA-mediated differentiation in stem cells and in certain cancers.

This study shows that Dis3l2 is the 3′–5′ exonuclease that mediates the degradation of uridylated precursor let-7 microRNA; this is the first physiological RNA substrate identified for this new exonuclease, which causes the Perlman syndrome of fetal overgrowth and Wilms’ tumour susceptibility when mutated. Function of Perlman syndrome exonuclease The Lin28-let-7 signalling cascade has been linked to stem cell function, cancers and various aspects of cellular metabolism. The pluripotency factor Lin28 recruits the 3′ terminal uridylyl transferases that add an oligouridine tail to let-7 precursor RNA. Here Richard Gregory and colleagues demonstrate that Dis3l2, a protein mutated in Perlman syndrome of fetal overgrowth and predisposition to Wilms' tumour, is the exonuclease that mediates the degradation of uridylated pre-let-7 in mouse embryonic stem cells. The identification of a decay pathway for uridylated RNAs raises the possibility that this type of post-transcriptional regulation might occur more widely. The pluripotency factor Lin28 blocks the expression of let-7 microRNAs in undifferentiated cells during development, and functions as an oncogene in a subset of cancers 1 . Lin28 binds to let-7 precursor (pre-let-7) RNAs and recruits 3′ terminal uridylyl transferases to selectively inhibit let-7 biogenesis 2 , 3 , 4 . Uridylated pre-let-7 is refractory to processing by Dicer, and is rapidly degraded by an unknown RNase 5 . Here we identify Dis3l2 as the 3′–5′ exonuclease responsible for the decay of uridylated pre-let-7 in mouse embryonic stem cells. Biochemical reconstitution assays show that 3′ oligouridylation stimulates Dis3l2 activity in vitro , and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3′ oligouridylation as an RNA decay signal for Dis3l2, and identifies the first physiological RNA substrate of this new exonuclease, which is mutated in the Perlman syndrome of fetal overgrowth and causes a predisposition to Wilms’ tumour development 6 .

Chemical modifications of RNA have key roles in many biological processes 1 – 3 . N 7 -methylguanosine (m 7 G) is required for integrity and stability of a large subset of tRNAs 4 – 7 . The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types 8 – 14 . Mutations in WDR4 cause human developmental phenotypes including microcephaly 15 – 17 . How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive 18 . Here we show,  through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity. Structures of the human METTL1–WDR4 complex are revealed, providing molecular insights into substrate recognition, modification and catalytic regulation by the N 7 -methylguanosine methyltransferase complex.

The let-7 microRNA has been implicated in development and disease. Its expression must thus be tightly regulated, and previously uridylation and Lin28 were implicated in let-7 stability. Zcchc11 is now shown to be the uridylase that mediates pre–let-7 modification and regulates mature let-7 levels and activity in mouse embryonic stem cells. Lin28 and Lin28B, two developmentally regulated RNA-binding proteins and likely proto-oncogenes, selectively inhibit the maturation of let-7 family microRNAs (miRNAs) in embryonic stem cells and certain cancer cell lines. Moreover, let-7 precursors (pre–let-7) were previously found to be terminally uridylated in a Lin28-dependent fashion. Here we identify Zcchc11 (zinc finger, CCHC domain containing 11) as the 3′ terminal uridylyl transferase (TUTase) responsible for Lin28-mediated pre–let-7 uridylation and subsequent blockade of let-7 processing in mouse embryonic stem cells. We demonstrate that Zcchc11 activity is UTP-dependent, selective for let-7 and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 miRNAs and repression of let-7 target reporter genes. Our results establish a role for Zcchc11-catalyzed pre–let-7 uridylation in the control of miRNA biogenesis.

The epitranscriptome includes a diversity of RNA modifications that influence gene expression. N3-methylcytidine (m 3 C) mainly occurs in the anticodon loop (position C32) of certain tRNAs yet its role is poorly understood. Here, using HAC-Seq, we report comprehensive METTL2A/2B-, METTL6-, and METTL2A/2B/6-dependent m 3 C profiles in human cells. METTL2A/2B modifies tRNA-arginine and tRNA-threonine members, whereas METTL6 modifies the tRNA-serine family. However, decreased m 3 C32 on tRNA-Ser-GCT isodecoders is only observed with combined METTL2A/2B/6 deletion. Ribo-Seq reveals altered translation of genes related to cell cycle and DNA repair pathways in METTL2A/2B/6-deficient cells, and these mRNAs are enriched in AGU codons that require tRNA-Ser-GCT for translation. These results, supported by reporter assays, help explain the observed altered cell cycle, slowed proliferation, and increased cisplatin sensitivity phenotypes of METTL2A/2B/6-deficient cells. Thus, we define METTL2A/2B/6-dependent methylomes and uncover a particular requirement of m 3 C32 tRNA modification for serine codon-biased mRNA translation of cell cycle, and DNA repair genes. Here the authors define METTL2A/2B/6-dependent m 3 C tRNA modification in human cells and uncover a particular requirement of m 3 C32 tRNA modification for serine codon-biased mRNA translation of cell cycle, and DNA repair genes.

Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

Background Adenosine deaminases acting on RNA (ADARs) modify many cellular RNAs by catalyzing the conversion of adenosine to inosine (A-to-I), and their deregulation is associated with several cancers. We recently showed that A-to-I editing is elevated in thyroid tumors and that ADAR1 is functionally important for thyroid cancer cell progression. The downstream effectors regulated or edited by ADAR1 and the significance of ADAR1 deregulation in thyroid cancer remain, however, poorly defined. Methods We performed whole transcriptome sequencing to determine the consequences of ADAR1 deregulation for global gene expression, RNA splicing and editing. The effects of gene silencing or RNA editing were investigated by analyzing cell viability, proliferation, invasion and subnuclear localization, and by protein and gene expression analysis. Results We report an oncogenic function for CDK13 in thyroid cancer and identify a new ADAR1-dependent RNA editing event that occurs in the coding region of its transcript. CDK13 was significantly over-edited (c.308A > G) in tumor samples and functional analysis revealed that this editing event promoted cancer cell hallmarks. Finally, we show that CDK13 editing increases the nucleolar abundance of the protein, and that this event might explain, at least partly, the global change in splicing produced by ADAR1 deregulation. Conclusions Overall, our data support A-to-I editing as an important pathway in cancer progression and highlight novel mechanisms that might be used therapeutically in thyroid and other cancers.

Key Points MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate target gene expression through mRNA degradation or translational inhibition. The miRNA biogenesis pathway is a multi-step process that has a crucial role in regulating miRNA maturation. miRNAs can be oncogenes or tumour suppressors and are globally repressed in cancers. Mutations in or dysregulation of components of the miRNA biogenesis pathway are frequently found in cancers and have important functions in oncogenesis. Important oncogenic signalling proteins — such as LIN28A, LIN28B, epidermal growth factor receptor (EGFR) and Hippo — target miRNA biogenesis in cancers. The targeting of abnormal miRNA biogenesis pathways is a novel, promising therapeutic strategy for cancers. The microRNA (miRNA) biogenesis pathway is frequently altered in cancer, leading to global downregulation of miRNA levels in some cancer types. This Review discusses the alterations that affect miRNA biogenesis in cancer. MicroRNAs (miRNAs) are critical regulators of gene expression. Amplification and overexpression of individual 'oncomiRs' or genetic loss of tumour suppressor miRNAs are associated with human cancer and are sufficient to drive tumorigenesis in mouse models. Furthermore, global miRNA depletion caused by genetic and epigenetic alterations in components of the miRNA biogenesis machinery is oncogenic. This, together with the recent identification of novel miRNA regulatory factors and pathways, highlights the importance of miRNA dysregulation in cancer.