The chromatin-associated lncREST ensures effective replication stress response by promoting the assembly of fork signaling factors.

Besides the well-characterized protein network involved in the replication stress response, several regulatory RNAs have been shown to play a role in this critical process. However, it has remained elusive whether they act locally at the stressed forks. Here, by investigating the RNAs localizing on chromatin upon replication stress induced by hydroxyurea, we identified a set of lncRNAs upregulated in S-phase and controlled by stress transcription factors. Among them, we demonstrate that the previously uncharacterized lncRNA lncREST (long non-coding RNA REplication STress) is transcriptionally controlled by p53 and localizes at stressed replication forks. LncREST-depleted cells experience sustained replication fork progression and accumulate un-signaled DNA damage. Under replication stress, lncREST interacts with the protein NCL and assists in engaging its interaction with RPA. The loss of lncREST is associated with a reduced NCL-RPA interaction and decreased RPA on chromatin, leading to defective replication stress signaling and accumulation of mitotic defects, resulting in apoptosis and a reduction in tumorigenic potential of cancer cells. These findings uncover the function of a lncRNA in favoring the recruitment of replication proteins to sites of DNA replication.

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

Revisiting the outcome of adult wild-type Htt inactivation in the context of HTT-lowering strategies for Huntington's disease.

Huntingtin-lowering strategies are central to therapeutic approaches for Huntington's disease. Recent studies reported the induction of age- and cell type-specific phenotypes by conditional huntingtin knockout, but these experimental conditions did not precisely mimic huntingtin-lowering or gene-editing conditions in terms of the cells targeted and brain distribution, and no transcriptional profiles were provided. Here, we used the adeno-associated delivery system commonly used in CNS gene therapy programmes and the self-inactivating KamiCas9 gene-editing system to investigate the long-term consequences of wild-type mouse huntingtin inactivation in adult neurons and, thus, the feasibility and safety of huntingtin inactivation in these cells. Behavioural and neuropathological analyses and single-nuclei RNA sequencing indicated that huntingtin editing in 77% of striatal neurons and 16% of cortical projecting neurons in adult mice induced no behavioural deficits or cellular toxicity. Single-nuclei RNA sequencing in 11.5-month-old animals showed that huntingtin inactivation did not alter striatal-cell profiles or proportions. Few differentially expressed genes were identified and Augur analysis confirmed an extremely limited response to huntingtin inactivation in all cell types. Our results therefore indicate that wild-type huntingtin inactivation in adult striatal and projection neurons is well tolerated in the long term.

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.

Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma.

Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1-cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

Gene regulation by long non-coding RNAs and its biological functions.

Evidence accumulated over the past decade shows that long non-coding RNAs (lncRNAs) are widely expressed and have key roles in gene regulation. Recent studies have begun to unravel how the biogenesis of lncRNAs is distinct from that of mRNAs and is linked with their specific subcellular localizations and functions. Depending on their localization and their specific interactions with DNA, RNA and proteins, lncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs and interfere with signalling pathways. Many of these functions ultimately affect gene expression in diverse biological and physiopathological contexts, such as in neuronal disorders, immune responses and cancer. Tissue-specific and condition-specific expression patterns suggest that lncRNAs are potential biomarkers and provide a rationale to target them clinically. In this Review, we discuss the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications.

Subcellular Distribution of p53 by the p53-Responsive lncRNA NBAT1 Determines Chemotherapeutic Response in Neuroblastoma.

Neuroblastoma has a low mutation rate for the p53 gene. Alternative ways of p53 inactivation have been proposed in neuroblastoma, such as abnormal cytoplasmic accumulation of wild-type p53. However, mechanisms leading to p53 inactivation via cytoplasmic accumulation are not well investigated. Here we show that the neuroblastoma risk-associated locus 6p22.3-derived tumor suppressor NBAT1 is a p53-responsive lncRNA that regulates p53 subcellular levels. Low expression of NBAT1 provided resistance to genotoxic drugs by promoting p53 accumulation in cytoplasm and loss from mitochondrial and nuclear compartments. Depletion of NBAT1 altered CRM1 function and contributed to the loss of p53-dependent nuclear gene expression during genotoxic drug treatment. CRM1 inhibition rescued p53-dependent nuclear functions and sensitized NBAT1-depleted cells to genotoxic drugs. Combined inhibition of CRM1 and MDM2 was even more effective in sensitizing aggressive neuroblastoma cells with p53 cytoplasmic accumulation. Thus, our mechanistic studies uncover an NBAT1-dependent CRM1/MDM2-based potential combination therapy for patients with high-risk neuroblastoma. SIGNIFICANCE: This study shows how a p53-responsive lncRNA mediates chemotherapeutic response by modulating nuclear p53 pathways and identifies a potential treatment strategy for patients with high-risk neuroblastoma.

Analysis of copy number alterations reveals the lncRNA ALAL-1 as a regulator of lung cancer immune evasion.

Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified lncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNFα- and NF-κB-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation.

A lncRNA-SWI/SNF complex crosstalk controls transcriptional activation at specific promoter regions.

LncRNAs have been shown to be direct players in chromatin regulation, but little is known about their role at active genomic loci. We investigate the role of lncRNAs in gene activation by profiling the RNA interactome of SMARCB1-containing SWI/SNF complexes in proliferating and senescent conditions. The isolation of SMARCB1-associated transcripts, together with chromatin profiling, shows prevalent association to active regions where SMARCB1 differentially binds locally transcribed RNAs. We identify SWINGN, a lncRNA interacting with SMARCB1 exclusively in proliferating conditions, exerting a pro-oncogenic role in some tumor types. SWINGN is transcribed from an enhancer and modulates the activation of GAS6 oncogene as part of a topologically organized region, as well as a larger network of pro-oncogenic genes by favoring SMARCB1 binding. Our results indicate that SWINGN influences the ability of the SWI/SNF complexes to drive epigenetic activation of specific promoters, suggesting a SWI/SNF-RNA cooperation to achieve optimal transcriptional activation.

lncRNA-DNA hybrids regulate distant genes.

lncRNAs actively regulate gene expression. They contribute to chromosomal interactions at close or distant genomic regions, which, in turn, regulate transcription [1]. Ariel et al [2] reveal in a recent study a new molecular mechanism of the Arabidopsis lncRNA APOLO. The authors extend previously reported functions of APOLO in cis-regulation of chromosomal looping and transcription of its neighbor gene to a set of distant genes involved in auxin-induced molecular pathways controlling lateral root development. Noteworthy, APOLO recognition of multiple trans-modulated targets occurs through a novel mechanism involving R-loop formation.

Correction to: SNHG15 is a bifunctional MYC-regulated noncoding locus encoding a lncRNA that promotes cell proliferation, invasion and drug resistance in colorectal cancer by interacting with AIF.

In the original publication of this article,[1] the Funding section needs to be revised, and the corrected Funding section appears below.

SNHG15 is a bifunctional MYC-regulated noncoding locus encoding a lncRNA that promotes cell proliferation, invasion and drug resistance in colorectal cancer by interacting with AIF.

BACKGROUND: Thousands of long noncoding RNAs (lncRNAs) are aberrantly expressed in various types of cancers, however our understanding of their role in the disease is still very limited. METHODS: We applied RNAseq analysis from patient-derived data with validation in independent cohort of patients. We followed these studies with gene regulation analysis as well as experimental dissection of the role of the identified lncRNA by multiple in vitro and in vivo methods. RESULTS: We analyzed RNA-seq data from tumors of 456 CRC patients compared to normal samples, and identified SNHG15 as a potentially oncogenic lncRNA that encodes a snoRNA in one of its introns. The processed SNHG15 is overexpressed in CRC tumors and its expression is highly correlated with poor survival of patients. Interestingly, SNHG15 is more highly expressed in tumors with high levels of MYC expression, while MYC protein binds to two E-box motifs on SNHG15 sequence, indicating that SNHG15 transcription is directly regulated by the oncogene MYC. The depletion of SNHG15 by siRNA or CRISPR-Cas9 inhibits cell proliferation and invasion, decreases colony formation as well as the tumorigenic capacity of CRC cells, whereas its overexpression leads to opposite effects. Gene expression analysis performed upon SNHG15 inhibition showed changes in multiple relevant genes implicated in cancer progression, including MYC, NRAS, BAG3 or ERBB3. Several of these genes are functionally related to AIF, a protein that we found to specifically interact with SNHG15, suggesting that the SNHG15 acts, at least in part, by regulating the activity of AIF. Interestingly, ROS levels, which are directly regulated by AIF, show a significant reduction in SNHG15-depleted cells. Moreover, knockdown of SNHG15 increases the sensitiveness of the cells to 5-FU, while its overexpression renders them more resistant to the chemotherapeutic drug. CONCLUSION: Altogether, these results describe an important role of SNHG15 in promoting colon cancer and mediating drug resistance, suggesting its potential as prognostic marker and target for RNA-based therapies.

hCLE/RTRAF-HSPC117-DDX1-FAM98B: A New Cap-Binding Complex That Activates mRNA Translation.

hCLE/C14orf166/RTRAF, DDX1, and HSPC117 are components of cytoplasmic mRNA-transporting granules kinesin-associated in dendrites. They have also been found in cytoplasmic ribosome-containing RNA granules that transport specific mRNAs halted for translation until specific neuronal signals renders them accessible to the translation machinery. hCLE associates to DDX1, HSPC117, and FAM98B in HEK293T cells and all four proteins bind to cap analog-containing resins. Competition and elution experiments indicate that binding of hCLE complex to cap resins is independent of eIF4E; the cap-binding factor needed for translation. Purified hCLE free of its associated proteins binds cap with low affinity suggesting that its interacting proteins modulate its cap association. hCLE silencing reduces hCLE accumulation and that of its interacting proteins and decreases mRNA translation. hCLE-associated RNAs have been isolated and sequenced; RNAs involved in mRNA translation are specifically associated. The data suggest that RNA granules may co-transport RNAs encoding proteins involved in specific functions together with RNAs that encode proteins needed for the translation of these specific RNAs and indicate an important role for hCLE modulating mRNA translation.

PR-LncRNA signature regulates glioma cell activity through expression of SOX factors.

Long non-coding RNAs (LncRNAs) have emerged as a relevant class of genome regulators involved in a broad range of biological processes and with important roles in tumor initiation and malignant progression. We have previously identified a p53-regulated tumor suppressor signature of LncRNAs (PR-LncRNAs) in colorectal cancer. Our aim was to identify the expression and function of this signature in gliomas. We found that the expression of the four PR-LncRNAs tested was high in human low-grade glioma samples and diminished with increasing grade of disease, being the lowest in glioblastoma samples. Functional assays demonstrated that PR-LncRNA silencing increased glioma cell proliferation and oncosphere formation. Mechanistically, we found an inverse correlation between PR-LncRNA expression and SOX1, SOX2 and SOX9 stem cell factors in human glioma biopsies and in glioma cells in vitro. Moreover, knock-down of SOX activity abolished the effect of PR-LncRNA silencing in glioma cell activity. In conclusion, our results demonstrate that the expression and function of PR-LncRNAs are significantly altered in gliomagenesis and that their activity is mediated by SOX factors. These results may provide important insights into the mechanisms responsible for glioblastoma pathogenesis.