Human antigen R regulates autophagic flux by stabilizing autophagy-associated mRNA in calcific aortic valve disease.

AIMS: The incidence of calcific aortic valve disease (CAVD) has risen over the last decade and is expected to continue rising; however, pharmacological approaches have proven ineffective. In this study, we evaluated the role and underlying mechanisms of human antigen R (HuR)-mediated post-transcriptional regulation in CAVD. METHODS AND RESULTS: We found that HuR was significantly upregulated in human calcified aortic valves and primary aortic valvular interstitial cells (VICs) following osteogenic stimulation. Subsequent functional studies revealed that HuR silencing ameliorated calcification both in vitro and in vivo. For the first time, we demonstrated that HuR directly interacted with the transcript of phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A), which mediates phosphatidylinositol signalling, facilitates autophagy, and acts as an mRNA stabilizer. HuR positively modulated PIP4K2A expression at the post-transcriptional level and consequently influenced the AKT/mTOR/ATG13 pathway to regulate autophagy and CAVD progression. CONCLUSION: Our study provides new insights into the post-transcriptional regulatory role of HuR in modulating autophagy-positive factors to regulate the pathogenesis of CAVD. Our findings highlight the potential of HuR as an innovative therapeutic target in CAVD treatment.

Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.

HYL1-CLEAVAGE SUBTILASE 1 (HCS1) suppresses miRNA biogenesis in response to light-to-dark transition.

The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway.

9-Aminoacridine Inhibits Ribosome Biogenesis by Targeting Both Transcription and Processing of Ribosomal RNA.

Aminoacridines, used for decades as antiseptic and antiparasitic agents, are prospective candidates for therapeutic repurposing and new drug development. Although the mechanisms behind their biological effects are not fully elucidated, they are most often attributed to the acridines' ability to intercalate into DNA. Here, we characterized the effects of 9-aminoacridine (9AA) on pre-rRNA metabolism in cultured mammalian cells. Our results demonstrate that 9AA inhibits both transcription of the ribosomal RNA precursors (pre-rRNA) and processing of the already synthesized pre-rRNAs, thereby rapidly abolishing ribosome biogenesis. Using a fluorescent intercalator displacement assay, we further show that 9AA can bind to RNA in vitro, which likely contributes to its ability to inhibit post-transcriptional steps in pre-rRNA maturation. These findings extend the arsenal of small-molecule compounds that can be used to block ribosome biogenesis in mammalian cells and have implications for the pharmacological development of new ribosome biogenesis inhibitors.

Coordinated post-transcriptional control of oncogene-induced senescence by UNR/CSDE1.

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.

Nuclear Protein Condensates and Their Properties in Regulation of Gene Expression.

Our understanding of the spatiotemporal regulation of eukaryotic gene expression has recently been greatly stimulated by the findings that many of the regulators of chromatin, transcription, and RNA processing form biomolecular condensates often assembled through liquid-liquid phase separation. Increasing number of reports suggest that these condensates functionally regulate gene expression, largely by concentrating the relevant biomolecules in the liquid-like micro-compartments. However, it remains poorly understood how the physicochemical properties, especially the material properties, of the condensates regulate gene expression activity. In this review, we discuss current data on various nuclear condensates and their biophysical properties with the underlying molecular interactions, and how they may functionally impact gene expression at the level of chromatin organization and activities, transcription, and RNA processing.

Regulation of translation by site-specific ribosomal RNA methylation.

Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms and can arise from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2'-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signaling pathways, such as MYC oncogene expression. Ablation of one prominent methylation resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2'-O-methylation can give rise to ribosomes with specialized function. This suggests a broader mechanism where the specific regulation of rRNA modification patterns fine tunes translation.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing.

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host's immune response in different virus-host combinations.

Post-transcriptional diversity in riboproteins and RNAs in aging and cancer.

Post-transcriptional (PtscM) and post-translational (PtrnM) modifications of nucleotides and amino acids are covalent modifications able to change physio-chemical properties of RNAs and proteins. In the ribosome, the adequate assembly of rRNAs and ribosomal protein subunits in the nucleolus ensures suitable translational activity, with protein synthesis tuned according to intracellular demands of energy production, replication, proliferation, and growth. Disruption in the regulatory control of PtscM and PtrnM can impair ribosome biogenesis and ribosome function. Ribosomal impairment may, in turn, impact the synthesis of proteins engaged in functions as varied as telomere maintenance, apoptosis, and DNA repair, as well as intersect with mitochondria and telomerase activity. These cellular processes often malfunction in carcinogenesis and senescence. Here we discuss regulatory mechanisms of PtscMs and PtrnMs on ribosomal function. We also address chemical modification in rRNAs and their impacts on cellular metabolism, replication control, and senescence. Further, we highlight similarities and differences of PtscMs and PtrnMs in ribosomal intermediates during aging and carcinogenesis. Understanding these regulatory mechanisms may uncover critical steps for the development of more efficient oncologic and anti-aging therapies.

Complex Conformational Dynamics of the Heart Failure-Associated Pre-miRNA-377 Hairpin Revealed by Single-Molecule Optical Tweezers.

Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.

The m6A RNA Modification Quantity and mRNA Expression Level of RNA Methylation-Related Genes in Head and Neck Squamous Cell Carcinoma Cell Lines and Patients.

RNA methylation at the nitrogen sixth of adenosine (m6A, N6-methyladenosine) is the most abundant RNA modification which plays a crucial role in all RNA metabolic aspects. Recently, m6A modification has been assigned to mediate the biological processes of cancer cells, but their significance in HNSCC development is still poorly described. Thus, the main aim of this study was to globally quantify m6A modification by the mass spectrometry approach and determine the mRNA expression level of selected m6A RNA methyltransferase (METTL3), demethylase (FTO), and m6A readers (YTHDF2, YTHDC2) in 45 HNSCC patients and 4 cell lines (FaDu, Detroit 562, A-253 and SCC-15) using qPCR. In the results, we have not observed differences in the global amount of m6A modification and the mRNA level of the selected genes between the cancerous and paired-matched histopathologically unchanged tissues from 45 HNSCC patients. However, we have found a positive correlation between selected RNA methylation machinery genes expression and m6A abundance on total RNA and characterized the transcript level of those genes in the HNSCC cell lines. Moreover, the lack of global m6A differences between cancerous and histopathologically unchanged tissues suggests that m6A alterations in specific RNA sites may specifically influence HNSCC tumorigenesis.

Interactions between m6A modification and miRNAs in malignant tumors.

Recently, the regulatory role of epigenetic modifications in the occurrence and development of malignant tumors has attracted extensive attention. RNA m6A methylation is the most abundant RNA modification in eukaryotic cells and regulates RNA transcription, processing, splicing, degradation, and translation. As important biomarkers, miRNAs play a crucial role in the diagnosis and treatment of diseases as well as in the development of anti-tumor drugs. Recently, increasing evidence has shown that m6A modification plays a vital role in regulating miRNA biosynthesis. We, herein, have reviewed the enzyme system involved in m6A methylation and the crosstalk between m6A modification and miRNAs in cancer. In addition, we have discussed the potential clinical applications and possible development directions of this field in the future.

MODOMICS: An Operational Guide to the Use of the RNA Modification Pathways Database.

MODOMICS is an established database of RNA modifications that provides comprehensive information concerning chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. This chapter covers the resources available on MODOMICS web server and the basic steps that can be undertaken by the user to explore them. MODOMICS is available at http://www.genesilico.pl/modomics .

Coronavirus genomic nsp14-ExoN, structure, role, mechanism, and potential application as a drug target.

The recent coronavirus disease 2019 (COVID-19), causing a global pandemic with devastating effects on healthcare and social-economic systems, has no special antiviral therapies available for human coronaviruses (CoVs). The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) possesses a nonstructural protein (nsp14), with amino-terminal domain coding for proofreading exoribonuclease (ExoN) that is required for high-fidelity replication. The ability of CoVs during genome replication and transcription to proofread and exclude mismatched nucleotides has long hindered the development of anti-CoV drugs. The resistance of SARS-CoV-2 to antivirals, especially nucleoside analogs (NAs), shows the need to identify new CoV inhibition targets. Therefore, this review highlights the importance of nsp14-ExoN as a target for inhibition. Also, nucleoside analogs could be used in combination with existing anti-CoV therapeutics to target the proofreading mechanism.

The Regulation of RNA Modification Systems: The Next Frontier in Epitranscriptomics?

RNA modifications, long considered to be molecular curiosities embellishing just abundant and non-coding RNAs, have now moved into the focus of both academic and applied research. Dedicated research efforts (epitranscriptomics) aim at deciphering the underlying principles by determining RNA modification landscapes and investigating the molecular mechanisms that establish, interpret and modulate the information potential of RNA beyond the combination of four canonical nucleotides. This has resulted in mapping various epitranscriptomes at high resolution and in cataloguing the effects caused by aberrant RNA modification circuitry. While the scope of the obtained insights has been complex and exciting, most of current epitranscriptomics appears to be stuck in the process of producing data, with very few efforts to disentangle cause from consequence when studying a specific RNA modification system. This article discusses various knowledge gaps in this field with the aim to raise one specific question: how are the enzymes regulated that dynamically install and modify RNA modifications? Furthermore, various technologies will be highlighted whose development and use might allow identifying specific and context-dependent regulators of epitranscriptomic mechanisms. Given the complexity of individual epitranscriptomes, determining their regulatory principles will become crucially important, especially when aiming at modifying specific aspects of an epitranscriptome both for experimental and, potentially, therapeutic purposes.

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo.

Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

The N6-methyladenosine modification posttranscriptionally regulates hepatic UGT2B7 expression.

UDP-glucuronosyltransferases (UGTs) are enzymes catalyzing the glucuronidation of various endogenous and exogenous compounds. In this study, we examined the possibility that N6-methyladenosine (m6A) modification affects hepatic UGT expression. Treatment of HepaRG cells with 3-deazaadenosine, an inhibitor of RNA methylation, significantly increased UGT1A1, UGT1A3, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B15 mRNA levels (1.3- to 2.6-fold). Among them, we focused on UGT2B7 because it most highly contributes to glucuronidation of clinically used drugs. Methylated RNA immunoprecipitation assays revealed that UGT2B7 mRNA in HepaRG cells and human livers is subjected to m6A modification mainly at the 5' untranslated region (UTR) and secondarily at the 3'UTR. UGT2B7 mRNA and protein levels in Huh-7 cells were significantly increased by double knockdown of methyltransferase-like 3 (METTL3) and METTL14, whereas those were decreased by knockdown of fat mass and obesity-associated protein (FTO) or alkB homolog 5, RNA demethylase (ALKBH5), suggesting that m6A modification downregulates UGT2B7 expression. By experiments using actinomycin D, an inhibitor of transcription, it was demonstrated that ALKBH5-mediated demethylation would attenuate UGT2B7 mRNA degradation, whereas METTL3/METTL14 or FTO-mediated m6A modification would alter the transactivity of UGT2B7. Luciferase assays revealed that the promoter region at -118 to -106 has a key role in the decrease in transactivity of UGT2B7 by FTO knockdown. We found that hepatocyte nuclear factor 4α (HNF4α) expression was significantly decreased by knockdown of FTO, indicating that this would be the underlying mechanism of the decreased transactivity of UGT2B7 by knockdown of FTO. Interestingly, treatment with entacapone, which is used for the treatment of Parkinson's disease and is an inhibitor of FTO, decreased HNF4α and UGT2B7 expression. In conclusion, this study clarified that RNA methylation posttranscriptionally controls hepatic UGT2B7 expression.

Revealing nascent RNA processing dynamics with nano-COP.

During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.

The Role of RNA Modifications and RNA-modifying Proteins in Cancer Therapy and Drug Resistance.

The advent of new genome-wide sequencing technologies has uncovered abnormal RNA modifications and RNA editing in a variety of human cancers. The discovery of reversible RNA N6-methyladenosine (RNA: m6A) by fat mass and obesity-associated protein (FTO) demethylase has led to exponential publications on the pathophysiological functions of m6A and its corresponding RNA modifying proteins (RMPs) in the past decade. Some excellent reviews have summarized the recent progress in this field. Compared to the extent of research into RNA: m6A and DNA 5-methylcytosine (DNA: m5C), much less is known about other RNA modifications and their associated RMPs, such as the role of RNA: m5C and its RNA cytosine methyltransferases (RCMTs) in cancer therapy and drug resistance. In this review, we will summarize the recent progress surrounding the function, intramolecular distribution and subcellular localization of several major RNA modifications, including 5' cap N7-methylguanosine (m7G) and 2'-O-methylation (Nm), m6A, m5C, A-to-I editing, and the associated RMPs. We will then discuss dysregulation of those RNA modifications and RMPs in cancer and their role in cancer therapy and drug resistance.

Histone mRNA is subject to 3' uridylation and re-adenylation in Aspergillus nidulans.

The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.