Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood (original) (raw)
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Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resist...
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International Journal of Molecular Sciences
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Long noncoding RNAs (lncRNAs) were discovered in eukaryotes more than thirty years ago [1]. Recent advances in genomics have led to the discovery that lncRNAs are transcribed pervasively across the genome [2-5]. There are an increasing number of reports that identify lncRNAs whose expression is modulated during cell differentiation or in disease states. However, biological functions for the vast majority of them are yet to be determined. Here, we propose two ways to identify lncRNAs that have biological functions: To identify lncRNAs with dedicated preinitiation complexes (PICs), and to focus on those whose transcription is highly regulated.
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bioRxiv (Cold Spring Harbor Laboratory), 2021
Transcrip&on of the human genome yields mostly long non-coding RNAs (lncRNAs). Systema&c func&onal annota&on of lncRNAs is challenging due to their low expression level, cell type-speciFc occurrence, poor sequence conserva&on between orthologs, and lack of informa&on about RNA domains. Currently, 95% of human lncRNAs have no func&onal characteriza&on. Using chroma&n conforma&on and Cap Analysis of Gene Expression (CAGE) data in 18 human cell types, we systema&cally located genomic regions in spa&al proximity to lncRNA genes and iden&Fed func&onal clusters of interac&ng protein-coding genes, lncRNAs and enhancers. Using these clusters we provide a cell type-speciFc func&onal annota&on for 7,651 out of 14,198 (53.88%) lncRNAs. LncRNAs tend to have specialized roles in the cell type in which it is Frst expressed, and to incorporate more general func&ons as its expression is acquired by mul&ple cell types during evolu&on. By analyzing RNA-binding protein and RNA-chroma&n interac&on data in the context of the spa&al genomic interac&on map, we explored mechanisms by which these lncRNAs can act.
Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene signature based on the top screening hits correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patientderived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
Nucleic acids research, 2015
Long non-coding RNAs (lncRNAs) have emerged as critical regulators of genes at epigenetic, transcriptional and post-transcriptional levels, yet what genes are regulated by a specific lncRNA remains to be characterized. To assess the effects of the lncRNA on gene expression, an increasing number of researchers profiled the genome-wide or individual gene expression level change after knocking down or overexpressing the lncRNA. Herein, we describe a curated database named LncRNA2Target, which stores lncRNA-to-target genes and is publicly accessible at http://www.lncrna2target.org. A gene was considered as a target of a lncRNA if it is differentially expressed after the lncRNA knockdown or overexpression. LncRNA2Target provides a web interface through which its users can search for the targets of a particular lncRNA or for the lncRNAs that target a particular gene. Both search types are performed either by browsing a provided catalog of lncRNA names or by inserting lncRNA/target gene ID...
Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display
Nature Methods, 2015
Noncoding RNAs (ncRNAs) comprise an important class of regulatory molecules that mediate a vast array of biological processes. This broad functional capacity has also facilitated the design of artificial ncRNAs with novel functions. To further investigate and harness these capabilities, we developed CRISPR-Display ("CRISP-Disp"), a targeted localization method that uses Sp. Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that exogenous RNA domains can be functionally appended onto the CRISPR scaffold at multiple insertion points, allowing the construction of Cas9 complexes with protein-binding cassettes, artificial aptamers, pools of random sequences, and RNAs up to 4.8 kilobases in length, including natural lncRNAs. Unlike most existing CRISPR methods, CRISP-Disp allows simultaneous multiplexing of distinct functions at multiple targets, limited only by the number of available functional RNA motifs. We anticipate that this technology will provide a powerful method with which to ectopically localize functional RNAs and ribonucleoprotein (RNP) complexes at specified genomic loci. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
PLoS ONE, 2014
Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as proteincoding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.
Genes, 2020
Although thousands of mammalian long non-coding RNAs (lncRNAs) have been reported in the last decade, their functional annotation remains limited. A wet-lab approach to detect functions of a novel lncRNA usually includes its knockdown followed by RNA sequencing and identification of the deferentially expressed genes. However, identification of the molecular mechanism(s) used by the lncRNA to regulate its targets frequently becomes a challenge. Previously, we developed the ASSA algorithm that detects statistically significant inter-molecular RNA-RNA interactions. Here we designed a workflow that uses ASSA predictions to estimate the ability of an lncRNA to function via direct base pairing with the target transcripts (co- or post-transcriptionally). The workflow was applied to 300+ lncRNA knockdown experiments from the FANTOM6 pilot project producing statistically significant predictions for 71 unique lncRNAs (104 knockdowns). Surprisingly, the majority of these lncRNAs were likely to...
Role of lncRNAs in health and disease--size and shape matter
Briefings in Functional Genomics, 2014
Most of the mammalian genome including a large fraction of the non-protein coding transcripts has been shown to be transcribed. Studies related to these non-coding RNA molecules have predominantly focused on smaller molecules like microRNAs. In contrast, long non-coding RNAs (lncRNAs) have long been considered to be transcriptional noise. Accumulating evidence suggests that lncRNAs are involved in key cellular and developmental processes. Several critical questions regarding functions and properties of lncRNAs and their circular forms remain to be answered. Increasing evidence from high-throughput sequencing screens also suggests the involvement of lncRNAs in diseases such as cancer, although the underlying mechanisms still need to be elucidated. Here, we discuss the current state of research in the field of lncRNAs, questions that need to be addressed in light of recent genome-wide studies documenting the landscape of lncRNAs, their functional roles and involvement in diseases. We posit that with the availability of high-throughput data sets it is not only possible to improve methods for predicting lncRNAs but will also facilitate our ability to elucidate their functions and phenotypes by using integrative approaches.