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.

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. Regio et al. report that long-term inactivation of wild-type huntingtin in striatal and cortical projecting neurons from adult mice is well tolerated. Graphical Abstract

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 wildtype 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.

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 IncRNA 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.

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 IncRNA 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 TNF alpha- and NF-kappa B-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.

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.

BackgroundThousands 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.MethodsWe 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.ResultsWe 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.ConclusionAltogether, 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.

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

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 elF4E; 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.