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Signaling and post-transcriptional gene control are both critical for the regulation of pluripotency1,2, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein (RBP), has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA (miRNA) biogenesis and direct modulation of mRNA translation3. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells (PSCs), which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced LIN28’s effect on its direct mRNA targets, revealing a mechanism that uncouples LIN28’s let-7-dependent and independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naïve to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signaling, post-transcriptional gene control, and cell fate regulation.

The article discusses research studies on the use of micro ribonucleic acids (RNA) as an effective method for reprogramming somatic cells to become pluripotent stem cells. The studies identified the micro RNA target genes that can stimulate the formation of the stem cells as well as dissect the pathways and mechanisms underlying the reprogramming of the cells. According to the author, the findings of these studies can be challenged by ethical and political issues related to human embryonic stem cell and regenerative medicine.

The article discusses a study that examines the microRNA regulation of stem cell fate. The study shows that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from embryonic stem (ES) cells but have opposing functions during further differentiation into cardiac muscle progenitors. The results show that muscle-specific microRNAs repress nonmuscle genes to direct embryonic stem cell differentiation to mesodem and muscle.

Summary METTL3 is an RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through N 6 -methyladenosine (m 6 A) modification. Here we find that METTL3 promotes translation of certain mRNAs including epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ in human cancer cells. In contrast to current models that invoke m 6 A reader proteins downstream of nuclear METTL3, we find METTL3 associates with ribosomes and promotes translation in the cytoplasm. METTL3 depletion inhibits translation, and both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA. Mechanistically, METTL3 enhances mRNA translation through an interaction with the translation initiation machinery. METTL3 expression is elevated in lung adenocarcinoma and using both loss- and gain-of-function studies, we find that METTL3 promotes growth, survival, and invasion of human lung cancer cells. Our results uncover an important role of METTL3 in promoting translation of oncogenes in human lung cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

RNA is subject to a multitude of different chemical modifications that collectively represent the epitranscriptome. Individual RNA modifications including N6-methyladenosine (m 6 A) on mRNA play essential roles in the posttranscriptional control of gene expression. Recent technological advances have enabled the transcriptome-wide mapping of certain RNA modifications, to reveal their broad relevance and characteristic distribution patterns. However, convenient methods that enable the simultaneous mapping of multiple different RNA marks within the same sample are generally lacking. Here we present EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout that expands the scope of molecular recognition-based RNA modification detection to multiple targets, while providing relative quantification and enabling low RNA input. Measuring signal intensity against spike-in controls provides relative quantification, indicative of the RNA mod abundance at each locus. We report on changes in the modification status of HEK293T cells upon treatment with pharmacological inhibitors separately targeting METTL3, the dominant m 6 A writer enzyme, and the EIF4A3 component of the exon junction complex (EJC). The treatments resulted in decreased or increased m 6 A levels, respectively, without effect on inosine levels. Inhibiting the helicase activity of EIF4A3 and EIF4A3 knockdown both cause a significant increase of m 6 A sites near exon junctions, consistent with the previously reported role of EIF4A3 in shaping the m 6 A landscape. Thus, EpiPlex offers a reliable and convenient method for simultaneous mapping of multiple RNA modifications to facilitate epitranscriptome studies.

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m 7 G) 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 m 7 G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m 7 G-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., Competing Interests: Declaration of interests R.I.G. and F.J.S. are co-founders and scientific advisory board members of 28-7 Therapeutics. R.I.G. is a co-founder and scientific advisory board member of Theon Therapeutics. E.Y. is co-funded by STORM Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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 (m 7 G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m 7 G TRAC-Seq involves the treatment of size-selected (<200 nt) RNAs with the demethylase AlkB to remove major tRNA modifications, followed by sodium borohydride (NaBH 4 ) reduction of m 7 G 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 m 7 G 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 m 7 G sites in tRNAs. The m 7 G 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.

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m 6 A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m 6 A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNA miR ) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNA miR -containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNA miR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNA miR -containing vectors for RNAi-based therapy., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N 7 -methylguanosine (m 7 G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m 7 G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m 7 G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs is modified at a "RAGGU" motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs, implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m 7 G tRNA methylome and highlights its essential role in ESCs with links to human disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature ∼22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part of a newly identified regulatory mechanism controlling Microprocessor activity.

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature 22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part ofa newly identified regulatory mechanism controlling Microprocessor activity.

The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that have been implicated in controlling diverse pathways in a variety of organisms (1, 2). For posttranscriptional gene silencing, one strand of the miRNA is used to guide components of the RNA interference machinery, including Argonaute 2, to messenger RNAs (mRNAs) with complementary sequences (3, 4). Thus, targeted mRNAs are either cleaved by the endonuclease Argonaute 2 (5, 6), or protein synthesis is blocked by an as yet uncharacterized mechanism (7, 8). Genes encoding miRNAs are transcribed as long primary miRNAs (pri-miRNAs) that are sequentially processed by components of the nucleus and cytoplasm to yield a mature, approx 22-nucleotide (nt)-long miRNA (9). Two members of the ribonuclease (RNase) III endonuclease protein family, Drosha and Dicer, have been implicated in this two-step processing (10-13). To further our understanding of miRNA biogenesis and function it will be essential to identify the protein complexes involved. We were interested in defining the proteins required for the initial nuclear processing of pri-miRNAs to the approx 60- to 70-nt stem-loop intermediates known as precursor miRNAs (pre-miRNAs) (9, 10). This led to our identification of a protein complex we termed Microprocessor, which is necessary and sufficient for processing pri-miRNA to premiRNAs (14). The Microprocessor complex comprises Drosha and the double-stranded RNAbinding protein DiGeorge syndrome critical region 8 gene (DGCR8), which is deleted in DiGeorge syndrome (15, 16). In this chapter, we detail the methods used for the biochemical isolation and identification of the Microprocessor complex from human cells. We include a protocol for the in vitro analysis of pri-miRNA processing activity of the purified Microprocessor complex.