Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation.

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. The establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show that mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.

A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA.

RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. It remains unclear whether pseudouridylation of mRNA can be snoRNA-guided, bearing important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at relatively low efficiencies, and is constrained by mRNA localization, snoRNA expression levels and the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously shown to suppress translational termination. However, in this and follow-up experiments in human cells we observe no evidence for significant levels of readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, 'design rules', constraints and consequences of snoRNA-mediated pseudouridylation.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.

Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N1-methyladenosine (m1A), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m1A at single-nucleotide resolution. Within the cytosol, m1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m1A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m1A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m1A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m1A levels was adopted as a potential means of post-transcriptional regulation.

Misincorporation signatures for detecting modifications in mRNA: Not as simple as it sounds.

Post-transcriptional modification on mRNA has become a field of intense interest in recent years, and next-generation sequencing based technologies are constantly emerging to detect an increasing number of modifications at a transcriptome-wide level. Some of these approaches are based on identification of misincorporation events induced by reverse transcriptase at modified sites. Although conceptually trivial, sensitive and specific identification of such events is a challenge prone to a surprising number of artifacts, which can lead to substantially inflated estimates of the abundance of diverse modifications. Here we discuss the sources of some of these artifacts and delineate approaches to overcome them.

Two phases of mitogenic signaling unveil roles for p53 and EGR1 in elimination of inconsistent growth signals.

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

Epigenetic mechanisms underlie the crosstalk between growth factors and a steroid hormone.

Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and glucocorticoids (GCs) involves transcription factors like p53 and NF-κB, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethylated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.

A Chemical Signature for Cytidine Acetylation in RNA.

N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease.

Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor.

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes.

The short and the long: non-coding RNAs and growth factors in cancer progression.

A relatively well-understood multistep process enables mutation-bearing cells to form primary tumours, which later use the circulation system to colonize new locations and form metastases. However, in which way the emerging abundance of different non-coding RNAs supports tumour progression is poorly understood. Here, we review new lines of evidence linking long and short types of non-coding RNAs to signalling pathways activated in the course of cancer progression by growth factors and by the tumour micro-environment. Resolving the new dimension of non-coding RNAs in oncogenesis will probably translate to earlier detection of cancer and improved therapeutic strategies.

Role of RNA modifications in cancer metastasis.

The epitranscriptome encompasses over 170 post-transcriptional modifications found in various RNA species. RNA modifications play pivotal roles in regulating gene expression by shaping RNA structure and function, implicating the epitranscriptome in diverse biological processes, including pathology progression. This review focuses on research elucidating the roles of the epitranscriptome in cancer metastasis. Metastasis, a primary cause of solid tumor patient mortality, involves a multistep process whereby tumor cells migrate from a primary tumor to distant secondary organs. We discuss RNA modifications found on rRNA, tRNA, and mRNA, highlighting their roles in different stages of metastasis. Understanding mechanisms by which modifications regulate molecular and cellular processes during metastasis is crucial for leveraging epitranscriptomic signatures in cancer diagnosis and treatment.

A crossroad of microRNAs and immediate early genes (IEGs) encoding oncogenic transcription factors in breast cancer.

Signaling networks are involved in development, as well as in malignancy of the mammary gland. Distinct external stimuli activate intricate signaling cascades, which culminate in the activation of specific transcriptional programs. These signal-specific transcriptional programs are instigated by transcription factors (TFs) encoded by the immediate early genes (IEGs), and they lead to diverse cellular outcomes, including oncogenesis. Hence, regulating the expression of IEGs is of great importance, and involves several complementary transcriptional and posttranscriptional mechanisms, the latter entails also microRNAs (miRNAs). miRNAs are a class of non-coding RNAs, which have been implicated in regulation of various aspects of signaling networks. Through examination of the basic characteristics of miRNA function, we highlight the benefits of using miRNAs as regulators of early TFs and signaling networks. We further focus on the role of miRNAs as regulators of IEGs, which shape the initial steps of signaling-induced transcription. We especially emphasize the role of miRNAs in buffering external noise and maintaining low basal activation of IEGs in the absence of proper stimuli.

A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit.

Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2'-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis.

mito-Ψ-Seq: A High-Throughput Method for Systematic Mapping of Pseudouridine Within Mitochondrial RNA.

RNA modifications are present in most cellular RNAs and are formed posttranscriptionally by enzymatic machineries that involve hundreds of enzymes and cofactors. RNA modifications impact the life cycle of the RNA, its stability, folding, cellular localization, as well as interactions with RNA and protein partners. RNA modifications are important for mitochondrial function and are required for proper processing and function of mitochondrial (mt) tRNA and rRNA. Underscoring their importance, several mitochondrial diseases are caused by defects in mt-RNA modifications, stemming from mutations in mtDNA at or near mt-RNA modification sites or in nuclear-encoded mt-RNA modifying enzymes. A highly abundant RNA modification, involved in mitochondrial physiology and pathology is pseudouridylation (Ψ), which is catalyzed by enzymes of the Pseudouridine Synthase (PUS) family. Although some Ψ sites in mt-rRNA and mt-tRNA have been identified, little is known about the functional role of these modifications. Furthermore, it is unknown which enzyme facilitates the modification of each site and it is likely that many yet undiscovered mt-RNA modifications exist, as is evidenced by recent work showing some Ψ sites on mRNA. Here, we present mito-Ψ-Seq, a high-throughput method for semiquantitative mapping of Ψ in mt-RNA.

Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq.

A prerequisite to defining the transcriptome-wide functions of RNA modifications is the ability to accurately determine their location. Here, we present N4-acetylcytidine (ac4C) sequencing (ac4C-seq), a protocol for the quantitative single-nucleotide resolution mapping of cytidine acetylation in RNA. This method exploits the kinetically facile chemical reaction of ac4C with sodium cyanoborohydride under acidic conditions to form a reduced nucleobase. RNA is then fragmented, ligated to an adapter at its 3' end and reverse transcribed to introduce a non-cognate nucleotide at reduced ac4C sites. After adapter ligation, library preparation and high-throughput sequencing, a bioinformatic pipeline enables identification of ac4C positions on the basis of the presence of C→T misincorporations in reduced samples but not in controls. Unlike antibody-based approaches, ac4C-seq identifies specific ac4C residues and reports on their level of modification. The ac4C-seq library preparation protocol can be completed in ~4 d for transcriptome-wide sequencing.

Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing.

Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N6-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA.

SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors.

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.

LIMT is a novel metastasis inhibiting lncRNA suppressed by EGF and downregulated in aggressive breast cancer.

Long noncoding RNAs (lncRNAs) are emerging as regulators of gene expression in pathogenesis, including cancer. Recently, lncRNAs have been implicated in progression of specific subtypes of breast cancer. One aggressive, basal-like subtype associates with increased EGFR signaling, while another, the HER2-enriched subtype, engages a kin of EGFR Based on the premise that EGFR-regulated lncRNAs might control the aggressiveness of basal-like tumors, we identified multiple EGFR-inducible lncRNAs in basal-like normal cells and overlaid them with the transcriptomes of over 3,000 breast cancer patients. This led to the identification of 11 prognostic lncRNAs. Functional analyses of this group uncovered LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT), a highly conserved lncRNA, which is depleted in basal-like and in HER2-positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. We also find that LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo In conclusion, lncRNAs dynamically regulated by growth factors might act as novel drivers of cancer progression and serve as prognostic biomarkers.

MicroRNAs and Growth Factors: An Alliance Propelling Tumor Progression.

Tumor progression requires cancer cell proliferation, migration, invasion, and attraction of blood and lymph vessels. These processes are tightly regulated by growth factors and their intracellular signaling pathways, which culminate in transcriptional programs. Hence, oncogenic mutations often capture growth factor signaling, and drugs able to intercept the underlying biochemical routes might retard cancer spread. Along with messenger RNAs, microRNAs play regulatory roles in growth factor signaling and in tumor progression. Because growth factors regulate abundance of certain microRNAs and the latter modulate the abundance of proteins necessary for growth factor signaling, the two classes of molecules form a dense web of interactions, which are dominated by a few recurring modules. We review specific examples of the alliance formed by growth factors and microRNAs and refer primarily to the epidermal growth factor (EGF) pathway. Clinical applications of the crosstalk between microRNAs and growth factors are described, including relevance to cancer therapy and to emergence of resistance to specific drugs.