Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

CAG repeat expansion in the Huntington's disease gene shapes linear and circular RNAs biogenesis.

Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.

Alterations of ribosomal RNA pseudouridylation in human breast cancer.

RNA modifications are key regulatory factors for several biological and pathological processes. They are abundantly represented on ribosomal RNA (rRNA), where they contribute to regulate ribosomal function in mRNA translation. Altered RNA modification pathways have been linked to tumorigenesis as well as to other human diseases. In this study we quantitatively evaluated the site-specific pseudouridylation pattern in rRNA in breast cancer samples exploiting the RBS-Seq technique involving RNA bisulfite treatment coupled with a new NGS approach. We found a wide variability among patients at different sites. The most dysregulated positions in tumors turned out to be hypermodified with respect to a reference RNA. As for 2'O-methylation level of rRNA modification, we detected variable and stable pseudouridine sites, with the most stable sites being the most evolutionary conserved. We also observed that pseudouridylation levels at specific sites are related to some clinical and bio-pathological tumor features and they are able to distinguish different patient clusters. This study is the first example of the contribution that newly available high-throughput approaches for site specific pseudouridine detection can provide to the understanding of the intrinsic ribosomal changes occurring in human tumors.

Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5'-3'exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.

QUADRatlas: the RNA G-quadruplex and RG4-binding proteins database.

RNA G-quadruplexes (RG4s) are non-canonical, disease-associated post-transcriptional regulators of gene expression whose functions are driven by RNA-binding proteins (RBPs). Being able to explore transcriptome-wide RG4 formation and interaction with RBPs is thus paramount to understanding how they are regulated and exploiting them as potential therapeutic targets. Towards this goal, we present QUADRatlas (https://rg4db.cibio.unitn.it), a database of experimentally-derived and computationally predicted RG4s in the human transcriptome, enriched with biological function and disease associations. As RBPs are key to their function, we mined known interactions of RG4s with such proteins, complemented with an extensive RBP binding sites dataset. Users can thus intersect RG4s with their potential regulators and effectors, enabling the formulation of novel hypotheses on RG4 regulation, function and pathogenicity. To support this capability, we provide analysis tools for predicting whether an RBP can bind RG4s, RG4 enrichment in a gene set, and de novo RG4 prediction. Genome-browser and table views allow exploring, filtering, and downloading the data quickly for individual genes and in batch. QUADRatlas is a significant step forward in our ability to understand the biology of RG4s, offering unmatched data content and enabling the integrated analysis of RG4s and their interactions with RBPs.

CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in 'regulation of RNA splicing' and 'mRNA catabolic process'. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6-Methyladenosine-Modified RNAs.

YTHDF proteins bind the N 6-methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition.

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

Human dyskerin binds to cytoplasmic H/ACA-box-containing transcripts affecting nuclear hormone receptor dependence.

BACKGROUND: Dyskerin is a nuclear protein involved in H/ACA box snoRNA-guided uridine modification of RNA. In humans, its defective function is associated with cancer development and induces specific post-transcriptional alterations of gene expression. In this study, we seek to unbiasedly identify mRNAs regulated by dyskerin in human breast cancer-derived cells. RESULTS: We find that dyskerin depletion affects the expression and the association with polysomes of selected mRNA isoforms characterized by the retention of H/ACA box snoRNA-containing introns. These snoRNA retaining transcripts (snoRTs) are bound by dyskerin in the cytoplasm in the form of shorter 3' snoRT fragments. We then characterize the whole cytoplasmic dyskerin RNA interactome and find both H/ACA box snoRTs and protein-coding transcripts which may be targeted by the snoRTs' guide properties. Since a fraction of these protein-coding transcripts is involved in the nuclear hormone receptor binding, we test to see if this specific activity is affected by dyskerin. Obtained results indicate that dyskerin dysregulation may alter the dependence on nuclear hormone receptor ligands in breast cancer cells. These results are paralleled by consistent observations on the outcome of primary breast cancer patients stratified according to their tumor hormonal status. Accordingly, experiments in nude mice show that the reduction of dyskerin levels in estrogen-dependent cells favors xenograft development in the absence of estrogen supplementation. CONCLUSIONS: Our work suggests a cytoplasmic function for dyskerin which could affect mRNA post-transcriptional networks relevant for nuclear hormone receptor functions.

Correction to: Post-Transcriptional Gene Regulation.

The book was inadvertently published with incorrect affiliation for the authors "Katherine B. Henke, Rachel M. Miller, Rachel A. Knoener, Mark Scalf, Michele Spiniello, and Lloyd M. Smith" in the contributors' list in FM and in the metadata of chapter 12.

A Community Study of SARS-CoV-2 Detection by RT-PCR in Saliva: A Reliable and Effective Method.

Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.

Introduction to Bioinformatics Resources for Post-transcriptional Regulation of Gene Expression.

Untranslated regions of mRNA (UTRs) are involved in defining the fate of the transcript through processes such as mRNA localization, degradation, translation initiation regulation, and several others: the action of trans-factors such as RNA-binding proteins and non-coding RNAs, combined with the presence of defined sequence and structural cis-elements, ultimately determines protein synthesis levels. Identifying functional regions in UTRs and uncovering post-transcriptional regulators acting upon these is thus of paramount importance to understand this regulatory layer: these tasks can now be approached computationally to reduce the testable hypothesis space and drive the experimental validation in a more effective way.This chapter will focus on presenting databases and tools allowing to study the various aspects of post-transcriptional regulation, including the profiling of actively translated mRNAs, regulatory network analysis (e.g., RBP and ncRNA binding sites), trans-factor binding sites prediction, motif search (sequence and secondary structure), and other aspects of this regulatory layer: two potential analysis pipelines are also presented as practical examples of how these tools could be integrated and effectively employed.

MODOMICS: a database of RNA modification pathways. 2021 update.

The MODOMICS database has been, since 2006, a manually curated and centralized resource, storing and distributing comprehensive information about modified ribonucleosides. Originally, it only contained data on the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. Over the years, prompted by the accumulation of new knowledge and new types of data, it has been updated with new information and functionalities. In this new release, we have created a catalog of RNA modifications linked to human diseases, e.g., due to mutations in genes encoding modification enzymes. MODOMICS has been linked extensively to RCSB Protein Data Bank, and sequences of experimentally determined RNA structures with modified residues have been added. This expansion was accompanied by including nucleotide 5'-monophosphate residues. We redesigned the web interface and upgraded the database backend. In addition, a search engine for chemically similar modified residues has been included that can be queried by SMILES codes or by drawing chemical molecules. Finally, previously available datasets of modified residues, biosynthetic pathways, and RNA-modifying enzymes have been updated. Overall, we provide users with a new, enhanced, and restyled tool for research on RNA modification. MODOMICS is available at https://iimcb.genesilico.pl/modomics/.

DHX30 Coordinates Cytoplasmic Translation and Mitochondrial Function Contributing to Cancer Cell Survival.

DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here, we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, the depletion of both DHX30 isoforms leads to higher global translation but slower proliferation and lower mitochondrial energy metabolism. Isoform-specific silencing supports a role for cytoplasmic DHX30 in modulating global translation. The impact on translation and proliferation was confirmed in U2OS and MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified fourteen mitoribosome transcripts we propose as direct DHX30 targets that can be used to explore the prognostic value of this mechanism in cancer. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.

Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma.

The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance. In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation. We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics. Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models. Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy. In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines. Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion. Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.

Multilayer and MATR3-dependent regulation of mRNAs maintains pluripotency in human induced pluripotent stem cells.

Matrin3 (MATR3) is a nuclear RNA/DNA-binding protein that plays pleiotropic roles in gene expression regulation by directly stabilizing target RNAs and supporting the activity of transcription factors by modulating chromatin architecture. MATR3 is involved in the differentiation of neural cells, and, here, we elucidate its critical functions in regulating pluripotent circuits in human induced pluripotent stem cells (hiPSCs). MATR3 downregulation affects hiPSCs' differentiation potential by altering key pluripotency regulators' expression levels, including OCT4, NANOG, and LIN28A by pleiotropic mechanisms. MATR3 binds to the OCT4 and YTHDF1 promoters favoring their expression. YTHDF1, in turn, binds the m6A-modified OCT4 mRNA. Furthermore, MATR3 is recruited on ribosomes and controls pluripotency regulating the translation of specific transcripts, including NANOG and LIN28A, by direct binding and favoring their stabilization. These results show that MATR3 orchestrates the pluripotency circuitry by regulating the transcription, translational efficiency, and epitranscriptome of specific transcripts.

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

A mark of disease: how mRNA modifications shape genetic and acquired pathologies.

RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). In mRNAs the deposition, removal, and recognition of chemical marks by writers, erasers and readers influence their structure, localization, stability, and translation. In turn, this modulates key molecular and cellular processes such as RNA metabolism, cell cycle, apoptosis, and others. Unsurprisingly, given their relevance for cellular and organismal functions, alterations of epitranscriptomic marks have been observed in a broad range of human diseases, including cancer, neurological and metabolic disorders. Here, we will review the major types of mRNA modifications and editing processes in conjunction with the enzymes involved in their metabolism and describe their impact on human diseases. We present the current knowledge in an updated catalog. We will also discuss the emerging evidence on the crosstalk of epitranscriptomic marks and what this interplay could imply for the dynamics of mRNA modifications. Understanding how this complex regulatory layer can affect the course of human pathologies will ultimately lead to its exploitation toward novel epitranscriptomic therapeutic strategies.