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RNA's ability to form and interconvert between multiple secondary and tertiary structures is critical to its functional versatility and the traditional view of RNA structures as static entities has shifted towards understanding them as dynamic conformational ensembles. In this review we discuss RNA structural ensembles and their dynamics, highlighting the concept of conformational energy landscapes as a unifying framework for understanding RNA processes such as folding, misfolding, conformational changes, and complex formation. Ongoing advancements in cryo-electron microscopy and chemical probing techniques are significantly enhancing our ability to investigate multiple structures adopted by conformationally dynamic RNAs, while traditional methods such as nuclear magnetic resonance spectroscopy continue to play a crucial role in providing high-resolution, quantitative spatial and temporal information. We discuss how these methods, when used synergistically, can provide a comprehensive understanding of RNA conformational ensembles, offering new insights into their regulatory functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Conformational dynamics is crucial for the biological function of RNA molecules and for their potential as therapeutic targets. This meeting report outlines key "take-home" messages that emerged from the presentations and discussions during the CECAM workshop "RNA dynamics from experimental and computational approaches" in Paris, June 26-28, 2023., Competing Interests: Declaration of interests P.G. and Y.F. are or were Sanofi employees and may own stocks in Sanofi., (Copyright © 2024.)

RNA molecules perform a variety of functions in cells, many of which rely on their secondary and tertiary structures. Chemical probing methods coupled with high-throughput sequencing have significantly accelerated the mapping of RNA structures, and increasingly large datasets of transcriptome-wide RNA chemical probing data are becoming available. Analogously to what has been done for decades in the protein world, this RNA structural information can be leveraged to aid the discovery of structural similarity to a known RNA (or RNA family), which, in turn, can inform about the function of transcripts. We have previously developed SHAPEwarp, a sequence-agnostic method for the search of structurally homologous RNA segments in a database of reactivity profiles derived from chemical probing experiments. In its original implementation, however, SHAPEwarp required substantial computational resources, even for moderately sized databases, as well as significant Linux command line know-how. To address these limitations, we introduce here SHAPEwarp-web, a user-friendly web interface to rapidly query large databases of RNA chemical probing data for structurally similar RNAs. Aside from featuring a completely rewritten core, which speeds up by orders of magnitude the search inside large databases, the web server hosts several high-quality chemical probing databases across multiple species. SHAPEwarp-web is available from https://shapewarp.incarnatolab.com., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. The 5' untranslated region (UTR) (5' UTR) of the NRAS mRNA contains a G-quadruplex (G4) that regulates translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5' UTR of the NRAS mRNA. We used a small molecule microarray screen to identify molecules that selectively bind to the NRAS-G4 with submicromolar affinity. One compound inhibits the translation of NRAS in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends and RT-PCR analysis revealed that the predominant NRAS transcript does not possess the G4 structure. Thus, although NRAS transcripts lack a G4 in many cell lines the concept of targeting folded regions within 5' UTRs to control translation remains a highly attractive strategy., Competing Interests: Author contributions S.B., Z.R.T., D.R.C., K.Y., X.L., C.R.F., and S.S. performed screening, biochemical, biological, and synthetic chemistry experiments described in the manuscript and helped write/edit the manuscript. M.B. performed X-ray crystallographic experiments and helped edit the manuscript. R.J.H. and T.A. performed and analyzed proteomics assays. A.R.F. edited the manuscript. D.I. performed SHAPE and CAGE experiments and edited the manuscript. J.S.S. conceived experiments and wrote/edited the manuscript. Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of β-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of β-Catenin depends on its ability to localize in and stabilize the β-Catenin destruction complex, promoting enhanced β-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the β-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response., (© 2023. The Author(s).)

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m 6 A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities., Competing Interests: Declaration of interests C.B. and S.M. are founders and members of the scientific advisory board of SACRA Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome - the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies., (© 2022. Springer Nature Limited.)

The identification of pseudo- and N 1 -methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues has propelled the development of mRNA-based vaccines and therapeutics. Here, we have characterised another uridine analogue, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it does not induce interleukin-6 secretion or expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression, as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU-modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity to the other uridine analogues. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent of RNA folding. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination., (© 2023. The Author(s).)

Chemical probing of RNA 2'-hydroxyl groups by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a rapid and powerful approach for querying RNA structures in living cells. At reverse transcription, sites of chemical modification can be encoded as mutations in the cDNA, a process called mutational profiling (MaP), enabling their detection via high-throughput sequencing. This chapter describes how to synthesize the SHAPE probe 2-aminopyridine-3-carboxylic acid imidazolide (2A3), how to use it to probe RNA structures in living bacteria, and how to generate Illumina-compatible SHAPE-MaP sequencing libraries. The protocol further describes data analysis using the RNA Framework, from raw sequencing data processing to experimentally-driven RNA secondary structure model generation., (Copyright © 2023. Published by Elsevier Inc.)

RNA structure probing experiments have emerged over the last decade as a straightforward way to determine the structure of RNA molecules in a number of different contexts. Although powerful, the ability of RNA to dynamically interconvert between, and to simultaneously populate, alternative structural configurations, poses a nontrivial challenge to the interpretation of data derived from these experiments. Recent efforts aimed at developing computational methods for the reconstruction of coexisting alternative RNA conformations from structure probing data are paving the way to the study of RNA structure ensembles, even in the context of living cells. In this review, we critically discuss these methods, their limitations and possible future improvements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4 + T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gigliotti, Boggio, Favero, Incarnato, Santoro, Oliviero, Rojo, Zucchelli, Persichetti, Baldanzi, Dianzani and Corà.)

The next generation sequencing has significantly contributed to clarify the genome structure of many species of zootechnical interest. However, to date, some portions of the genome, especially those linked to a heterogametic nature such as the Y chromosome, are difficult to assemble and many gaps are still present. It is well known that the fluorescence in situ hybridization (FISH) is an excellent tool for identifying genes unequivocably mapped on chromosomes. Therefore, FISH can contribute to the localization of unplaced genome sequences, as well as to correct assembly errors generated by comparative bioinformatics. To this end, it is necessary to have starting points; therefore, in this study, we reviewed the physically mapped genes on the Y chromosome of cattle, buffalo, sheep, goats, pigs, horses and alpacas. A total of 208 loci were currently mapped by FISH. 89 were located in the malespecific region of the Y chromosome (MSY) and 119 were identified in the pseudoautosomal region (PAR). The loci reported in MSY and PAR were respectively: 18 and 25 in Bos taurus, 5 and 7 in Bubalus bubalis, 5 and 24 in Ovis aries, 5 and 19 in Capra hircus, 10 and 16 in Sus scrofa, 46 and 18 in Equus caballus. While in Vicugna pacos only 10 loci are reported in the PAR region. The correct knowledge and assembly of all genome sequences, including those of genes mapped on the Y chromosome, will help to elucidate their biological processes, as well as to discover and exploit potentially epistasis effects useful for selection breeding programs.

Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function., Results: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina., Conclusions: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions., (© 2022. The Author(s).)

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes., (© 2022. The Author(s).)

The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear (1,2). We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the tumor microenvironment, Cancer Associated Fibroblasts (CAFs) become activated by cancer cells and increase their secretory activity to produce soluble factors that contribute to tumor cells proliferation, invasion and dissemination to distant organs. The pro-tumorigenic transcription factor STAT3 and its canonical inducer, the pro-inflammatory cytokine IL-6, act conjunctly in a positive feedback loop that maintains high levels of IL-6 secretion and STAT3 activation in both tumor and stromal cells. Here, we demonstrate that STAT3 is essential for the pro-tumorigenic functions of murine breast cancer CAFs both in vitro and in vivo, and identify a STAT3 signature significantly enriched for genes encoding for secreted proteins. Among these, ANGPTL4, MMP13 and STC-1 were functionally validated as STAT3-dependent mediators of CAF pro-tumorigenic functions by different approaches. Both in vitro and in vivo CAFs activities were moreover impaired by MMP13 inhibition, supporting the feasibility of a therapeutic approach based on inhibiting STAT3-induced CAF-secreted proteins. The clinical potential of such an approach is supported by the observation that an equivalent CAF-STAT3 signature in humans is expressed at high levels in breast cancer stromal cells and characterizes patients with a shorter disease specific survival, including those with basal-like disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)