Transmission of reduced levels of miR-34/449 from sperm to preimplantation embryos is a key step in the transgenerational epigenetic inheritance of the effects of paternal chronic social instability stress.

The transgenerational effects of exposing male mice to chronic social instability (CSI) stress are associated with decreased sperm levels of multiple members of the miR-34/449 family that persist after their mating through preimplantation embryo (PIE) development. Here we demonstrate the importance of these miRNA changes by showing that restoring miR-34c levels in PIEs derived from CSI stressed males prevents elevated anxiety and defective sociability normally found specifically in their adult female offspring. It also restores, at least partially, levels of sperm miR-34/449 normally reduced in their male offspring who transmit these sex-specific traits to their offspring. Strikingly, these experiments also revealed that inducing miR-34c levels in PIEs enhances the expression of its own gene and that of miR-449 in these cells. The same induction of embryo miR-34/449 gene expression likely occurs after sperm-derived miR-34c is introduced into oocytes upon fertilization. Thus, suppression of this miRNA amplification system when sperm miR-34c levels are reduced in CSI stressed mice can explain how a comparable fold-suppression of miR-34/449 levels can be found in PIEs derived from them, despite sperm containing ~50-fold lower levels of these miRNAs than those already present in PIEs. We previously found that men exposed to early life trauma also display reduced sperm levels of miR-34/449. And here we show that miR-34c can also increase the expression of its own gene, and that of miR-449 in human embryonic stem cells, suggesting that human PIEs derived from men with low sperm miR-34/449 levels may also contain this potentially harmful defect.

Benchmarking of computational methods for m6A profiling with Nanopore direct RNA sequencing.

N6-methyladenosine (m6A) is the most abundant internal eukaryotic mRNA modification, and is involved in the regulation of various biological processes. Direct Nanopore sequencing of native RNA (dRNA-seq) emerged as a leading approach for its identification. Several software were published for m6A detection and there is a strong need for independent studies benchmarking their performance on data from different species, and against various reference datasets. Moreover, a computational workflow is needed to streamline the execution of tools whose installation and execution remains complicated. We developed NanOlympicsMod, a Nextflow pipeline exploiting containerized technology for comparing 14 tools for m6A detection on dRNA-seq data. NanOlympicsMod was tested on dRNA-seq data generated from in vitro (un)modified synthetic oligos. The m6A hits returned by each tool were compared to the m6A position known by design of the oligos. In addition, NanOlympicsMod was used on dRNA-seq datasets from wild-type and m6A-depleted yeast, mouse and human, and each tool's hits were compared to reference m6A sets generated by leading orthogonal methods. The performance of the tools markedly differed across datasets, and methods adopting different approaches showed different preferences in terms of precision and recall. Changing the stringency cut-offs allowed for tuning the precision-recall trade-off towards user preferences. Finally, we determined that precision and recall of tools are markedly influenced by sequencing depth, and that additional sequencing would likely reveal additional m6A sites. Thanks to the possibility of including novel tools, NanOlympicsMod will streamline the benchmarking of m6A detection tools on dRNA-seq data, improving future RNA modification characterization.

Unveiling the role of PUS7-mediated pseudouridylation in host protein interactions specific for the SARS-CoV-2 RNA genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2 UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

Preimplantation Embryos Amplify Stress-Induced Changes in Sperm-Derived miRNA Content to Mediate Transgenerational Epigenetic Inheritance.

Chronically stressing male mice can alter the behavior of their offspring across generations. This effect is thought to be mediated by stress-induced changes in the content of specific sperm miRNAs that modify embryo development after their delivery to oocytes at fertilization. A major problem with this hypothesis is that the levels of mouse sperm miRNAs are much lower than those present in preimplantation embryos. This makes it unclear how embryos could be significantly impacted without an amplification system to magnify changes in sperm miRNA content, like those present in lower organisms where transgenerational epigenetic inheritance is well established. Here, we describe such a system for Chronic Social Instability (CSI) stress that can explain how it reduces the levels of the miR-34b,c/449a,b family of miRNAs not only in sperm of exposed males but also in preimplantation embryos ( PIEs ) derived from their mating, as well as in sperm of male offspring. Sperm-derived miR-34c normally positively regulates expression of its own gene and that of miR-449 in PIEs. This feed forward, auto-amplification process is suppressed when CSI stress reduces sperm miR-34c levels. Its suppression is important for the transmission of traits to offspring because restoring miR-34c levels in PIEs from CSI stressed males, which also restores levels of miR-449 in them, suppresses elements of elevated anxiety and defective sociability normally found specifically in their female offspring, as well as reduced sperm miR-34 and miR-449 levels normally found in male offspring, who pass on these traits to their offspring. We previously published that the content of sperm miR-34/449 is also reduced in men raised in highly abusive and/or dysfunctional families. We show here that a similar miRNA auto-amplification system functions in human embryonic stem cells. This raises the possibility that PIEs in offspring of these men also display reduced levels of miR-34/449, enhancing the potential translational significance of these studies.

iNKT cell-neutrophil crosstalk promotes colorectal cancer pathogenesis.

iNKT cells account for a relevant fraction of effector T-cells in the intestine and are considered an attractive platform for cancer immunotherapy. Although iNKT cells are cytotoxic lymphocytes, their functional role in colorectal cancer (CRC) is still controversial, limiting their therapeutic use. Thus, we examined the immune cell composition and iNKT cell phenotype of CRC lesions in patients (n = 118) and different murine models. High-dimensional single-cell flow-cytometry, metagenomics, and RNA sequencing experiments revealed that iNKT cells are enriched in tumor lesions. The tumor-associated pathobiont Fusobacterium nucleatum induces IL-17 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in iNKT cells without affecting their cytotoxic capability but promoting iNKT-mediated recruitment of neutrophils with polymorphonuclear myeloid-derived suppressor cells-like phenotype and functions. The lack of iNKT cells reduced the tumor burden and recruitment of immune suppressive neutrophils. iNKT cells in-vivo activation with α-galactosylceramide restored their anti-tumor function, suggesting that iNKT cells can be modulated to overcome CRC-associated immune evasion. Tumor co-infiltration by iNKT cells and neutrophils correlates with negative clinical outcomes, highlighting the importance of iNKT cells in the pathophysiology of CRC. Our results reveal a functional plasticity of iNKT cells in CRC, suggesting a pivotal role of iNKT cells in shaping the tumor microenvironment, with relevant implications for treatment.

Mechanisms of Long Non-Coding RNA in Breast Cancer.

The landscape of pervasive transcription in eukaryotic genomes has made space for the identification of thousands of transcripts that are difficult to frame in a specific functional category. A new class has been broadly named as long non-coding RNAs (lncRNAs) and shortly defined as transcripts that are longer than 200 nucleotides with no or limited coding potential. So far, about 19,000 lncRNAs genes have been annotated in the human genome (Gencode 41), nearly matching the number of protein-coding genes. A key scientific priority is the functional characterization of lncRNAs, a major challenge in molecular biology that has encouraged many high-throughput efforts. LncRNA studies have been stimulated by the enormous clinical potential that these molecules promise and have been based on the characterization of their expression and functional mechanisms. In this review, we illustrate some of these mechanisms as they have been pictured in the context of breast cancer.

Using Nanocompore to Identify RNA Modifications from Direct RNA Nanopore Sequencing Data.

RNA modifications can alter the behavior of RNA molecules depending on where they are located on the strands. Traditionally, RNA modifications have been detected and characterized by biophysical assays, mass spectrometry, or specific next-generation sequencing techniques, but are limited to specific modifications or are low throughput. Nanopore is a platform capable of sequencing RNA strands directly, which permits transcriptome-wide detection of RNA modifications. RNA modifications alter the nanopore raw signal relative to the canonical form of the nucleotide, and several software tools detect these signal alterations. One such tool is Nanocompore, which compares the ionic current features between two different experimental conditions (i.e., with and without RNA modifications) to detect RNA modifications. Nanocompore is not limited to a single type of RNA modification, has a high specificity for detecting RNA modifications, and does not require model training. To use Nanocompore, the following steps are needed: (i) the data must be basecalled and aligned to the reference transcriptome, then the raw ionic current signals are aligned to the sequences and transformed into a Nanocompore-compatible format; (ii) finally, the statistical testing is conducted on the transformed data and produces a table of p-value predictions for the positions of the RNA modifications. These steps can be executed with several different methods, and thus we have also included two alternative protocols for running Nanocompore. Once the positions of RNA modifications are determined by Nanocompore, users can investigate their function in various metabolic pathways. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: RNA modification detection by Nanocompore Alternate Protocol 1: RNA modification detection by Nanocompore with f5c Alternate Protocol 2: RNA modification detection by Nanocompore using Nextflow.

Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells.

Proximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a 20 nm range. These enzymes can be either fused to an RNA binding protein or tethered to any RNA of interest and expressed in living cells to biotinylate the amino acids and nucleic acids of binding partners in proximity. The biotinylated molecules can then be easily affinity purified under denaturing conditions and analyzed by mass spectrometry or next generation sequencing. These approaches have been widely used in recent years, providing a potent instrument to map the molecular interactions of specific RNA-binding proteins as well as RNA transcripts occurring in mammalian cells. In addition, they permit the identification of transient interactions as well as interactions among low expressed molecules that are often missed by standard affinity purification strategies. This review will provide a brief overview of the currently available proximity ligation methods, highlighting both their strengths and shortcomings. Furthermore, it will bring further insights to the way these technologies could be further used to characterize post-transcriptional modifications that are known to regulate RNA-protein interactions.

News from around the RNA world: new avenues in RNA biology, biotechnology and therapeutics from the 2022 SIBBM meeting.

Since the formalization of the Central Dogma of molecular biology, the relevance of RNA in modulating the flow of information from DNA to proteins has been clear. More recently, the discovery of a vast set of non-coding transcripts involved in crucial aspects of cellular biology has renewed the enthusiasm of the RNA community. Moreover, the remarkable impact of RNA therapies in facing the COVID19 pandemics has bolstered interest in the translational opportunities provided by this incredible molecule. For all these reasons, the Italian Society of Biophysics and Molecular Biology (SIBBM) decided to dedicate its 17th yearly meeting, held in June 2022 in Rome, to the many fascinating aspects of RNA biology. More than thirty national and international speakers covered the properties, modes of action and applications of RNA, from its role in the control of development and cell differentiation to its involvement in disease. Here, we summarize the scientific content of the conference, highlighting the take-home message of each presentation, and we stress the directions the community is currently exploring to push forward our comprehension of the RNA World 3.0.

microRNAs transcriptional profiling of mammary stem cells isolated by PKH26 staining and FACS sorting.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNA expression is frequently altered in human cancers compared to normal tissues, potentially contributing to tumorigenesis. Generally, high-throughput profiles of miRNA expression levels are generated using bulk samples, from both normal and cancer tissues. However, cancer tissues are quite heterogeneous and might contain subpopulations critical for tumor development, i.e., cancer stem cells (CSCs) or tumor-initiating cells (TICs) with aberrant stem-like features, such as unlimited self-renewal potential. The isolation of these aberrant subpopulations from solid tumors is a relatively recent achievement, with breast cancer being one of the first solid human cancers in which CSCs have been identified and biologically characterized. Here, we describe a new methodology that can overcome the main challenge in dealing with rare cells such as SCs/CSCs, represented by the paucity of the starting material. Based on previously published protocols, used by both our and other research groups, we used the FACS-sorting approach to isolate mammary normal and cancer stem cells based on the amount of PKH26 fluorescent dye they retained. Depending on the number of SCs/CSCs isolated, we established two different protocols for the reliable and analytically sensitive detection of up to 384 miRNAs using the Taqman Low Density Array (TLDA) platform.

A multifunctional locus controls motor neuron differentiation through short and long noncoding RNAs.

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.

Nanopore ReCappable sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

Prediction and pan-cancer analysis of mammalian transcripts involved in target directed miRNA degradation.

It is currently unknown how many RNA transcripts are able to induce degradation of microRNAs (miRNA) via the mechanism known as target-directed miRNA degradation (TDMD). We developed TDMDfinder, a computational pipeline that identifies 'high confidence' TDMD interactions in the Human and Mouse transcriptomes by combining sequence alignment and feature selection approaches. Our predictions suggested that TDMD is widespread, with potentially every miRNA controlled by endogenous targets. We experimentally tested 37 TDMDfinder predictions, of which 17 showed TDMD effects as measured by RT-qPCR and small RNA sequencing, linking the miR-17, miR-19, miR-30, miR-221, miR-26 and miR-23 families to novel endogenous TDMDs. In some cases, TDMD was found to affect different members of the same miRNA family selectively. Features like complementarity to the miRNA 3' region, bulge size and hybridization energy appeared to be the main factors determining sensitivity. Computational analyses performed using the multiomic TCGA platform substantiated the involvement of many TDMD transcripts in human cancer and highlighted 36 highly significant interactions, suggesting TDMD as a new potential oncogenic mechanism. In conclusion, TDMDfinder provides the first inventory of bona fide human and mouse TDMDs. Available as a free webtool, TDMDfinder allows users to search for any TDMD interaction of interest by customizing its selection criteria.

Microglia-specific overexpression of α-synuclein leads to severe dopaminergic neurodegeneration by phagocytic exhaustion and oxidative toxicity.

Recent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson's disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells.

miR-146 connects stem cell identity with metabolism and pharmacological resistance in breast cancer.

Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a "hidden vulnerability" exploitable for the development of anti-CSC therapies.

Frataxin gene editing rescues Friedreich's ataxia pathology in dorsal root ganglia organoid-derived sensory neurons.

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis.

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.

Correction: Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LncRNA EPR controls epithelial proliferation by coordinating Cdkn1a transcription and mRNA decay response to TGF-ß.

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-ß and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-ß.