Structural basis of regulated m7G tRNA modification by METTL1-WDR4.

Chemical modifications of RNA have key roles in many biological processes1-3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4-7. 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 types8-14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15-17. How METTL1-WDR4 modifies tRNA substrates and is regulated remains elusive18. 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.

Ribo-uORF: a comprehensive data resource of upstream open reading frames (uORFs) based on ribosome profiling.

Upstream open reading frames (uORFs) are typically defined as translation sites located within the 5' untranslated region upstream of the main protein coding sequence (CDS) of messenger RNAs (mRNAs). Although uORFs are prevalent in eukaryotic mRNAs and modulate the translation of downstream CDSs, a comprehensive resource for uORFs is currently lacking. We developed Ribo-uORF (http://rnainformatics.org.cn/RiboUORF) to serve as a comprehensive functional resource for uORF analysis based on ribosome profiling (Ribo-seq) data. Ribo-uORF currently supports six species: human, mouse, rat, zebrafish, fruit fly, and worm. Ribo-uORF includes 501 554 actively translated uORFs and 107 914 upstream translation initiation sites (uTIS), which were identified from 1495 Ribo-seq and 77 quantitative translation initiation sequencing (QTI-seq) datasets, respectively. We also developed mRNAbrowse to visualize items such as uORFs, cis-regulatory elements, genetic variations, eQTLs, GWAS-based associations, RNA modifications, and RNA editing. Ribo-uORF provides a very intuitive web interface for conveniently browsing, searching, and visualizing uORF data. Finally, uORFscan and UTR5var were developed in Ribo-uORF to precisely identify uORFs and analyze the influence of genetic mutations on uORFs using user-uploaded datasets. Ribo-uORF should greatly facilitate studies of uORFs and their roles in mRNA translation and posttranscriptional control of gene expression.

Exon junction complex shapes the m6A epitranscriptome.

N6-methyladenosine (m6A), the most abundant modification of mRNA, is essential for normal development and dysregulation promotes cancer. m6A 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 m6A distribution remains enigmatic. Here we find the exon junction complex shapes the m6A landscape by blocking METTL3-mediated m6A modification close to exon junctions within coding sequence (CDS). Depletion of EIF4A3, a core component of the EJC, causes increased METTL3 binding and m6A 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 m6A modification via the local steric blockade of METTL3. Our results explain how characteristic patterns of m6A mRNA modification are established and uncover a role of the EJC in shaping the m6A epitranscriptome.

tRNA dysregulation and disease.

tRNAs are key adaptor molecules that decipher the genetic code during translation of mRNAs in protein synthesis. In contrast to the traditional view of tRNAs as ubiquitously expressed housekeeping molecules, awareness is now growing that tRNA-encoding genes display tissue-specific and cell type-specific patterns of expression, and that tRNA gene expression and function are both dynamically regulated by post-transcriptional RNA modifications. Moreover, dysregulation of tRNAs, mediated by alterations in either their abundance or function, can have deleterious consequences that contribute to several distinct human diseases, including neurological disorders and cancer. Accumulating evidence shows that reprogramming of mRNA translation through altered tRNA activity can drive pathological processes in a codon-dependent manner. This Review considers the emerging evidence in support of the precise control of functional tRNA levels as an important regulatory mechanism that coordinates mRNA translation and protein expression in physiological cell homeostasis, and highlights key examples of human diseases that are linked directly to tRNA dysregulation.

LIN28B alters ribosomal dynamics to promote metastasis in MYCN-driven malignancy.

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

An ADAR1-dependent RNA editing event in the cyclin-dependent kinase CDK13 promotes thyroid cancer hallmarks.

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.

METTL1-mediated m7G modification of Arg-TCT tRNA drives oncogenic transformation.

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.

Genetic drivers of m6A methylation in human brain, lung, heart and muscle.

The most prevalent post-transcriptional mRNA modification, N6-methyladenosine (m6A), plays diverse RNA-regulatory roles, but its genetic control in human tissues remains uncharted. Here we report 129 transcriptome-wide m6A 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 m6A quantitative trait loci (QTLs), which are modestly enriched in, but mostly orthogonal to, expression QTLs. We integrate m6A QTLs with disease genetics, identifying 184 GWAS-colocalized m6A QTL, including brain m6A QTLs underlying neuroticism, depression, schizophrenia and anxiety; lung m6A QTLs underlying expiratory flow and asthma; and muscle/heart m6A QTLs underlying coronary artery disease. Last, we predict novel m6A regulators that show preferential binding in m6A QTLs, protein interactions with known m6A regulators and expression correlation with the m6A 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.

An Informatics Pipeline for Profiling and Annotating RNA Modifications.

While over 150 distinct types of chemical modifications are known to occur on various cellular RNAs and can be dynamically controlled, the function of most of these modifications remains poorly defined. Collectively, these RNA modifications have been recently termed the "epitranscriptome". Identification and annotation of individual RNA modifications throughout the transcriptome are key for studying the role of the epitranscriptome in the regulation of gene expression and for elucidating the functional relevance of particular RNA modifications in diverse physiological and disease processes. In this protocol, we demonstrate how to identify and annotate RNA modifications based on the informatic analysis of methylated RNA immunoprecipitation and sequencing (MeRIP-seq) data, using RNAmod, a convenient one-stop online interactive platform for the annotation, analysis, and visualization of mRNA modifications.

Global miRNA dosage control of embryonic germ layer specification.

MicroRNAs (miRNAs) have essential functions during embryonic development, and their dysregulation causes cancer1,2. Altered global miRNA abundance is found in different tissues and tumours, which implies that precise control of miRNA dosage is important1,3,4, but the underlying mechanism(s) of this control remain unknown. The protein complex Microprocessor, which comprises one DROSHA and two DGCR8 proteins, is essential for miRNA biogenesis5-7. Here we identify a developmentally regulated miRNA dosage control mechanism that involves alternative transcription initiation (ATI) of DGCR8. ATI occurs downstream of a stem-loop in DGCR8 mRNA to bypass an autoregulatory feedback loop during mouse embryonic stem (mES) cell differentiation. Deletion of the stem-loop causes imbalanced DGCR8:DROSHA protein stoichiometry that drives irreversible Microprocessor aggregation, reduced primary miRNA processing, decreased mature miRNA abundance, and widespread de-repression of lipid metabolic mRNA targets. Although global miRNA dosage control is not essential for mES cells to exit from pluripotency, its dysregulation alters lipid metabolic pathways and interferes with embryonic development by disrupting germ layer specification in vitro and in vivo. This miRNA dosage control mechanism is conserved in humans. Our results identify a promoter switch that balances Microprocessor autoregulation and aggregation to precisely control global miRNA dosage and govern stem cell fate decisions during early embryonic development.

Nucleotide resolution profiling of m3C RNA modification by HAC-seq.

Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called 'epitranscriptome', contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.

The mRNA m6A reader YTHDF2 suppresses proinflammatory pathways and sustains hematopoietic stem cell function.

The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

TRIM71 binds to IMP1 and is capable of positive and negative regulation of target RNAs.

TRIM71 is an important RNA-binding protein in development and disease, yet its direct targets have not been investigated globally. Here we describe a number of disease and developmentally-relevant TRIM71 RNA targets such as the MBNL family, LIN28B, MDM2, and TCF7L2. We describe a new role for TRIM71 as capable of positive or negative RNA regulation depending on the RNA target. We found that TRIM71 co-precipitated with IMP1 which could explain its multiple mechanisms of RNA regulation, as IMP1 is typically thought to stabilize RNAs. Deletion of the NHL domain of TRIM71 impacted its ability to bind to RNA and RNAs bound by congenital hydrocephalus-associated point mutations in the RNA-binding NHL domain of TRIM71 clustered closely with RNAs bound by the NHL deletion mutant. Our work expands the possible mechanisms by which TRIM71 may regulate RNAs and elucidates further potential RNA targets.

RiboToolkit: an integrated platform for analysis and annotation of ribosome profiling data to decode mRNA translation at codon resolution.

Ribosome profiling (Ribo-seq) is a powerful technology for globally monitoring RNA translation; ranging from codon occupancy profiling, identification of actively translated open reading frames (ORFs), to the quantification of translational efficiency under various physiological or experimental conditions. However, analyzing and decoding translation information from Ribo-seq data is not trivial. Although there are many existing tools to analyze Ribo-seq data, most of these tools are designed for specific or limited functionalities and an easy-to-use integrated tool to analyze Ribo-seq data is lacking. Fortunately, the small size (26-34 nt) of ribosome protected fragments (RPFs) in Ribo-seq and the relatively small amount of sequencing data greatly facilitates the development of such a web platform, which is easy to manipulate for users with or without bioinformatic expertise. Thus, we developed RiboToolkit (http://rnabioinfor.tch.harvard.edu/RiboToolkit), a convenient, freely available, web-based service to centralize Ribo-seq data analyses, including data cleaning and quality evaluation, expression analysis based on RPFs, codon occupancy, translation efficiency analysis, differential translation analysis, functional annotation, translation metagene analysis, and identification of actively translated ORFs. Besides, easy-to-use web interfaces were developed to facilitate data analysis and intuitively visualize results. Thus, RiboToolkit will greatly facilitate the study of mRNA translation based on ribosome profiling.

The Perlman syndrome DIS3L2 exoribonuclease safeguards endoplasmic reticulum-targeted mRNA translation and calcium ion homeostasis.

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.

Nucleotide resolution profiling of m7G tRNA modification by TRAC-Seq.

Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased global mapping of 7-methylguansine (m7G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m7G TRAC-Seq involves the treatment of size-selected (<200 nt) RNAs with the demethylase AlkB to remove major tRNA modifications, followed by sodium borohydride (NaBH4) reduction of m7G sites and subsequent aniline-mediated cleavage of the RNA chain at the resulting abasic sites. The cleaved sites are subsequently ligated with adaptors for the construction of libraries for high-throughput sequencing. The m7G modification sites are identified using a bioinformatic pipeline that calculates the cleavage scores at individual sites on all tRNAs. Unlike antibody-based methods, such as methylated RNA immunoprecipitation and sequencing (meRIP-Seq) for enrichment of methylated RNA sequences, chemically based approaches, including TRAC-Seq, can provide nucleotide-level resolution of modification sites. Compared to the related method AlkAniline-Seq (alkaline hydrolysis and aniline cleavage sequencing), TRAC-Seq incorporates small RNA selection, AlkB demethylation, and sodium borohydride reduction steps to achieve specific and efficient single-nucleotide resolution profiling of m7G sites in tRNAs. The m7G TRAC-Seq protocol could be adapted to chemical cleavage-mediated detection of other RNA modifications. The protocol can be completed within ~9 d for four biological replicates of input and treated samples.

RNAmod: an integrated system for the annotation of mRNA modifications.

Dynamic and reversible RNA modifications such as N6-methyladenosine (m6A) can play important roles in regulating messenger RNA (mRNA) splicing, export, stability and translation. Defective mRNA modification through altered expression of the methyltransferase and/or demethylases results in developmental defects and cancer progression. Identifying modified mRNAs, annotating the distribution of modification sites across the mRNA, as well as characterizing and comparing other modification features are essential for studying the function and elucidating the mechanism of mRNA modifications. Several methods including methylated RNA immunoprecipitation and sequencing (MeRIP-seq) are available for the detection of mRNA modifications. However, a convenient and comprehensive tool to annotate diverse kinds of mRNA modifications in different species is lacking. Here, we developed RNAmod (https://bioinformatics.sc.cn/RNAmod), an interactive, one-stop, web-based platform for the automated analysis, annotation, and visualization of mRNA modifications in 21 species. RNAmod provides intuitive interfaces to show outputs including the distribution of RNA modifications, modification coverage for different gene features, functional annotation of modified mRNAs, and comparisons between different groups or specific gene sets. Furthermore, sites of known RNA modification, as well as binding site data for hundreds of RNA-binding proteins (RBPs) are integrated in RNAmod to help users compare their modification data with known modifications and to explore the relationship with the binding sites of known RBPs. RNAmod is freely available and meets the emerging need for a convenient and comprehensive analysis tool for the fast-developing RNA modification field.