mRNA Regulation by RNA Modifications.

Chemical modifications on mRNA represent a critical layer of gene expression regulation. Research in this area has continued to accelerate over the last decade, as more modifications are being characterized with increasing depth and breadth. mRNA modifications have been demonstrated to influence nearly every step from the early phases of transcript synthesis in the nucleus through to their decay in the cytoplasm, but in many cases, the molecular mechanisms involved in these processes remain mysterious. Here, we highlight recent work that has elucidated the roles of mRNA modifications throughout the mRNA life cycle, describe gaps in our understanding and remaining open questions, and offer some forward-looking perspective on future directions in the field.

R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

Chemical Modifications in the Life of an mRNA Transcript.

Investigations over the past eight years of chemical modifications on messenger RNA (mRNA) have revealed a new level of posttranscriptional gene regulation in eukaryotes. Rapid progress in our understanding of these modifications, particularly, N6-methyladenosine (m6A), has revealed their roles throughout the life cycle of an mRNA transcript. m6A methylation provides a rapid mechanism for coordinated transcriptome processing and turnover that is important in embryonic development and cell differentiation. In response to cellular signals, m6A can also regulate the translation of specific pools of transcripts. These mechanisms can be hijacked in human diseases, including numerous cancers and viral infection. Beyond m6A, many other mRNA modifications have been mapped in the transcriptome, but much less is known about their biological functions. As methods continue to be developed, we will be able to study these modifications both more broadly and in greater depth, which will likely reveal a wealth of new RNA biology.

Enzymes flying under the radar: Cryptic METTL3 can persist in knockout cells.

The presence of m6A in mRNA of METTL3 knockout cells has long been a point of confusion. In this issue of PLOS Biology, Poh and colleagues reveal alternatively spliced, catalytically active METTL3 isoforms that persist in cells previously thought to lack the enzyme.

Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA.

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs.

RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Corrigendum: RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

This corrects the article DOI: 10.1038/nature21671.

Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA.

Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m1A sites and revealed hundreds of m1A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m1A biology.

The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Structural basis of Smoothened regulation by its extracellular domains.

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

The emerging biology of RNA post-transcriptional modifications.

RNA modifications have long been known to be central in the proper function of tRNA and rRNA. While chemical modifications in mRNA were discovered decades ago, their function has remained largely mysterious until recently. Using enrichment strategies coupled to next generation sequencing, multiple modifications have now been mapped on a transcriptome-wide scale in a variety of contexts. We now know that RNA modifications influence cell biology by many different mechanisms - by influencing RNA structure, by tuning interactions within the ribosome, and by recruiting specific binding proteins that intersect with other signaling pathways. They are also dynamic, changing in distribution or level in response to stresses such as heat shock and nutrient deprivation. Here, we provide an overview of recent themes that have emerged from the substantial progress that has been made in our understanding of chemical modifications across many major RNA classes in eukaryotes.

Tracking the subcellular fate of 20(s)-hydroxycholesterol with click chemistry reveals a transport pathway to the Golgi.

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.

Oxysterols are allosteric activators of the oncoprotein Smoothened.

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.

Combating the myth of "innate genius" in science.

In this Stories piece, Sigrid Nachtergaele-an assistant professor at the Yale University Department of Molecular, Cellular, and Developmental Biology-shares her experience and efforts to cut barriers for the next generation of aspiring scientists.

A user guide to RT-based mapping of RNA modifications.

Chemical modifications to RNA nucleotides are both a naturally occurring layer of biological regulation and an increasingly prevalent approach to synthetically alter RNA function in therapeutic applications. Detection of their presence, prevalence, and stoichiometry across different RNAs is critical to understanding their underlying functions. However, this remains challenging due to the technical barriers involved in differentiating chemically similar modification species, and in detecting rare or low stoichiometry modifications. Reverse transcription-based techniques rely on the introduction of a predictable mutation, truncation, or deletion signature when a reverse transcriptase encounters a modified nucleotide of interest. Previous studies have shown promise in detecting modifications to single nucleotide resolution, but the low efficiency and processivity of many commercially available reverse transcriptases has resulted in discordant conclusions in some cases. Here, we present guidelines and best practices for applying the highly processive MarathonRT enzyme to reverse transcription-based modification sequencing. These guidelines include recommendations for controls and example protocols to help users plan robust experiments for mapping modification(s) of choice, as well as discussion of the limitations for the methods described.

Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain.

Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.