Publications

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5'UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5'UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5'UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

Low expression levels and stoichiometric imbalances of long noncoding RNAs (lncRNAs) are often used as evidence for their probable lack of function or for limiting the scope of their potential influence. Recent advances in our understanding of the substoichiometric functions of lncRNAs challenge these notions and suggest routes through which unabundant lncRNAs can affect cellular functions and gene regulatory networks.

The synthesis of RNA polymerase II (Pol2) products, which include messenger RNAs or long noncoding RNAs, culminates in transcription termination. How the transcriptional termination of a gene impacts the activity of promoters found immediately downstream of it, and which can be subject to potential transcriptional interference, remains largely unknown. We examined in an unbiased manner the features of the intergenic regions between pairs of ‘tandem genes’—closely spaced (< 2 kb) human genes found on the same strand. Intergenic regions separating tandem genes are enriched with guanines and are characterized by binding of several proteins, including AGO1 and AGO2 of the RNA interference pathway. Additionally, we found that Pol2 is particularly enriched in this region, and it is lost upon perturbations affecting splicing or transcriptional elongation. Perturbations of genes involved in Pol2 pausing and R loop biology preferentially affect expression of downstream genes in tandem gene pairs. Overall, we find that features associated with Pol2 pausing and accumulation rather than those associated with avoidance of transcriptional interference are the predominant driving force shaping short tandem intergenic regions.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as β-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-β/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

Long noncoding RNAs (lncRNAs) have been shown to play important roles in gene regulatory networks acting in early development. There has been rapid turnover of lncRNA loci during vertebrate evolution, with few human lncRNAs conserved beyond mammals. The sequences of these rare deeply conserved lncRNAs are typically not similar to each other. Here, we characterize HOXA-AS3 and HOXB-AS3, lncRNAs produced from the central regions of the HOXA and HOXB clusters. Sequence-similar homologs of both lncRNAs are found in multiple vertebrate species and there is evident sequence similarity between their promoters, suggesting that the production of these lncRNAs predates the duplication of the HOX clusters at the root of the vertebrate lineage. This conservation extends to similar expression patterns of the two lncRNAs, in particular in cells transiently arising during early development or in the adult colon, and their co-regulation by the CDX1/2 transcription factors. Functionally, the RNA products of HOXA-AS3 and HOXB-AS3 regulate the expression of their overlapping HOX5-7 genes both in HT-29 cells and during differentiation of human embryonic stem cells. Beyond production of paralogous protein-coding and microRNA genes, the regulatory program in the HOX clusters therefore also relies on paralogous lncRNAs acting in restricted spatial and temporal windows of embryonic development and cell differentiation.

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+ /PSA+ ) or NEPC (AR- /SYN+ /CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease.

In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet, how transcriptional regulation of cell-cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle gene expression and increase in self-renewal gene expression are coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long noncoding RNA (lncRNA) that we named Stem Cell Inhibitory RNA Transcript (SCIRT), which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell-cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell-cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing toward repression. These data suggest that the interaction of an lncRNA with EZH2 can alter the affinity of EZH2 for its protein-binding partners to regulate cancer cell state transitions.

The auditory system is a complex sensory network with an orchestrated multilayer regulatory programme governing its development and maintenance. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) as important regulators in numerous systems, as well as in pathological pathways. However, their function in the auditory system has yet to be explored. Using a set of specific criteria, we selected four lncRNAs expressed in the mouse cochlea, which are conserved in the human transcriptome and are relevant for inner ear function. Bioinformatic characterization demonstrated a lack of coding potential and an absence of evolutionary conservation that represent properties commonly shared by their class members. RNAscope®  analysis of the spatial and temporal expression profiles revealed specific localization to inner ear cells. Sub-cellular localization analysis presented a distinct pattern for each lncRNA and mouse tissue expression evaluation displayed a large variability in terms of level and location. Our findings establish the expression of specific lncRNAs in different cell types of the auditory system and present a potential pathway by which the lncRNA Gas5 acts in the inner ear. Studying lncRNAs and deciphering their functions may deepen our knowledge of inner ear physiology and morphology and may reveal the basis of as yet unresolved genetic hearing loss-related pathologies. Moreover, our experimental design may be employed as a reference for studying other inner ear-related lncRNAs, as well as lncRNAs expressed in other sensory systems.

The nuclear export pathway transports long RNAs produced in the nucleus to the cytoplasm. The core components of this pathway are thought to be required for export of virtually all polyadenylated RNAs. Here, we depleted different proteins that act in nuclear export in human cells, and quantified the transcriptome-wide consequences on RNA localization. Different genes exhibited substantially variable sensitivities, with depletion of NXF1 and TREX components causing some transcripts to become strongly retained in the nucleus while others were not affected. Specifically, NXF1 is preferentially required for export of single- or few-exon transcripts with long exons or high A/U-content, whereas depletion of TREX complex components preferentially affects spliced and G/C-rich transcripts. Using massively parallel reporter assays we identified short sequence elements that render transcripts dependent on NXF1 for their export, and identified synergistic effects of splicing and NXF1. These results revise the current model of how nuclear export shapes the distribution of RNA within human cells.

Research on non-coding RNA (ncRNA) is a rapidly expanding field. Providing an official gene symbol and name to ncRNA genes brings order to otherwise potential chaos as it allows unambiguous communication about each gene. The HUGO Gene Nomenclature Committee (HGNC, www.genenames.org) is the only group with the authority to approve symbols for human genes. The HGNC works with specialist advisors for different classes of ncRNA to ensure that ncRNA nomenclature is accurate and informative, where possible. Here, we review each major class of ncRNA that is currently annotated in the human genome and describe how each class is assigned a standardised nomenclature.

Long noncoding RNAs (lncRNAs) are gathering increasing attention toward their roles in different biological systems. In mammals, the richest repertoires of lncRNAs are expressed in the brain and in the testis, and the diversity of lncRNAs in the nervous system is thought to be related to the diversity and the complexity of its cell types. Supporting this notion, many lncRNAs are differentially expressed between different regions of the brain or in particular cell types, and many lncRNAs are dynamically expressed during embryonic or postnatal neurogenesis. Less is known about the functions of these genes, if any, but they are increasingly implicated in diverse processes in health and disease. Here, we review the current knowledge about the roles and importance of lncRNAs in the central and peripheral nervous systems and discuss the specific niches within gene regulatory networks that might be preferentially occupied by lncRNAs.

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.

Present throughout the vasculature, endothelial cells (ECs) are essential for blood vessel function and play a central role in the pathogenesis of diverse cardiovascular diseases. Understanding the intricate molecular determinants governing endothelial function and dysfunction is essential to develop novel clinical breakthroughs and improve knowledge. An increasing body of evidence demonstrates that long non-coding RNAs (lncRNAs) are active regulators of the endothelial transcriptome and function, providing emerging insights into core questions surrounding EC contributions to pathology, and perhaps the emergence of novel therapeutic opportunities. In this review, we discuss this class of non-coding transcripts and their role in endothelial biology during cardiovascular development, homeostasis, and disease, highlighting challenges during discovery and characterization and how these have been overcome to date. We further discuss the translational therapeutic implications and the challenges within the field, highlighting lncRNA that support endothelial phenotypes prevalent in cardiovascular disease.

Genome-wide analysis of cellular transcriptomes using RNA-seq or expression arrays is a major mainstay of current biological and biomedical research. EXPANDER (EXPression ANalyzer and DisplayER) is a comprehensive software package for analysis of expression data, with built-in support for 18 different organisms. It is designed as a "one-stop shop" platform for transcriptomic analysis, allowing for execution of all analysis steps starting with gene expression data matrix. Analyses offered include low-level preprocessing and normalization, differential expression analysis, clustering, bi-clustering, supervised grouping, high-level functional and pathway enrichment tests, and networks and motif analyses. A variety of options is offered for each step, using established algorithms, including many developed and published by our laboratory. EXPANDER has been continuously developed since 2003, having to date over 18,000 downloads and 540 citations. One of the innovations in the recent version is support for combined analysis of gene expression and ChIP-seq data to enhance the inference of transcriptional networks and their functional interpretation. EXPANDER implements cutting-edge algorithms and makes them accessible to users through user-friendly interface and intuitive visualizations. It is freely available to users at http://acgt.cs.tau.ac.il/expander/.

The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs-alternative splicing and polyadenylation-have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3'UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

In mammals, neurons in the peripheral nervous system (PNS) have regenerative capacity following injury, but it is generally absent in the CNS. This difference is attributed, at least in part, to the intrinsic ability of PNS neurons to activate a unique regenerative transcriptional program following injury. Here, we profiled gene expression following sciatic nerve crush in mice and identified long noncoding RNAs (lncRNAs) that act in the regenerating neurons and which are typically not expressed in other contexts. We show that two of these lncRNAs regulate the extent of neuronal outgrowth. We then focus on one of these, Silc1, and show that it regulates neuroregeneration in cultured cells and in vivo, through cis-acting activation of the transcription factor Sox11.

Active enhancers in mammals produce enhancer RNAs (eRNAs), that are bidirectionally transcribed, unspliced, and unstable noncoding RNAs. Enhancer regions are also enriched with long noncoding RNA (lncRNA) genes, which are typically spliced and are longer and substantially more stable than eRNAs. In order to explore the relationship between these two classes of RNAs and the implications of lncRNA transcription on enhancer functionality, we analyzed DNAse hypersensitive sites with evidence of bidirectional transcription, which we termed eRNA producing centers (EPCs). A subset of EPCs, which are found very close to the transcription start site of lncRNA genes, exhibit attributes of both enhancers and promoters, including distinctive DNA motifs and a characteristic landscape of bound proteins. These EPCs are associated with a subset of relatively highly active enhancers. This stronger enhancer activity is driven, at least in part, by the presence of evolutionary conserved, directional splicing signals that promote lncRNA production, pointing at a causal role of lncRNA processing in enhancer activity. Together, our results suggest a model whereby the ability of some enhancers to produce lncRNAs, which is conserved in evolution, enhances their activity in a manner likely mediated through maturation of the associated lncRNA.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain’s macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the ‘RiboTag’ method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.

Long noncoding RNAs (lncRNAs) are emerging as key parts of multiple cellular pathways, but their modes of action and how these are dictated by sequence remain unclear. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic, although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus. It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. Here, to identify elements in lncRNAs and mRNAs that can force nuclear localization, we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increased nuclear accumulation. Binding of HNRNPK to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus identify a pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts with integrated Alu elements.