A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

CeFra-seq reveals broad asymmetric mRNA and noncoding RNA distribution profiles in Drosophila and human cells.

Cells are highly asymmetrical, a feature that relies on the sorting of molecular constituents, including proteins, lipids, and nucleic acids, to distinct subcellular locales. The localization of RNA molecules is an important layer of gene regulation required to modulate localized cellular activities, although its global prevalence remains unclear. We combine biochemical cell fractionation with RNA-sequencing (CeFra-seq) analysis to assess the prevalence and conservation of RNA asymmetric distribution on a transcriptome-wide scale in Drosophila and human cells. This approach reveals that the majority (∼80%) of cellular RNA species are asymmetrically distributed, whether considering coding or noncoding transcript populations, in patterns that are broadly conserved evolutionarily. Notably, a large number of Drosophila and human long noncoding RNAs and circular RNAs display enriched levels within specific cytoplasmic compartments, suggesting that these RNAs fulfill extra-nuclear functions. Moreover, fraction-specific mRNA populations exhibit distinctive sequence characteristics. Comparative analysis of mRNA fractionation profiles with that of their encoded proteins reveals a general lack of correlation in subcellular distribution, marked by strong cases of asymmetry. However, coincident distribution profiles are observed for mRNA/protein pairs related to a variety of functional protein modules, suggesting complex regulatory inputs of RNA localization to cellular organization.

CeFra-seq: Systematic mapping of RNA subcellular distribution properties through cell fractionation coupled to deep-sequencing.

The subcellular trafficking of RNA molecules is a conserved feature of eukaryotic cells and plays key functions in diverse processes implicating polarised cellular activities. Large-scale imaging and subcellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subcellular distribution properties. Here, we present a method combining biochemical fractionation of cells and high-throughput RNA sequencing (CeFra-seq) that enables rapid and efficient systematic mapping of RNA cytotopic distributions in cells. The described procedure involves biochemical fractionation to derive extracts of nuclear, cytosolic, endomembrane, cytoplasmic insoluble and extracellular material from cell culture lines. The RNA content of each fraction can then be profiled by deep-sequencing, revealing global subcellular signatures. We provide a detailed protocol for the CeFra-seq procedure along with relevant validation steps and data analysis guidelines to graphically represent RNA spatial distribution features. As a complement to imaging approaches, CeFra-seq represents a powerful and scalable tool to investigate global alterations in RNA trafficking.

The functions and regulatory principles of mRNA intracellular trafficking.

The subcellular localization of RNA molecules is a key step in the control of gene expression that impacts a broad array of biological processes in different organisms and cell types. Like other aspects of posttranscriptional gene regulation discussed in this collection of reviews, the intracellular trafficking of mRNAs is modulated by a complex regulatory code implicating specific cis-regulatory elements, RNA-binding proteins, and cofactors that function combinatorially to dictate precise localization mechanisms. In this review, we first discuss the functional benefits of transcript localization, the regulatory principles involved, and specific molecular mechanisms that have been described for a few well-characterized mRNAs. We also overview some of the emerging genomic and imaging technologies that have provided significant insights into this layer of gene regulation. Finally, we highlight examples of human diseases where defective transcript localization has been documented.

MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression.

Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK(+)) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK(+) ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK(+) ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.

Inter-dependent Centrosomal Co-localization of the cen and ik2 cis-Natural Antisense mRNAs in Drosophila.

Overlapping genes are prevalent in most genomes, but the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. Studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed as cis-natural antisense transcripts (cis-NATs) with overlapping 3' UTRs, we found that their encoded mRNAs strikingly co-localize to centrosomes. These transcripts physically interact in a 3' UTR-dependent manner, and the targeting of ik2 requires its 3' UTR sequence and the presence of cen mRNA, which serves as the main driver of centrosomal co-localization. The cen transcript undergoes localized translation in proximity to centrosomes, and its localization is perturbed by polysome-disrupting drugs. By interrogating global fractionation-sequencing datasets generated from Drosophila and human cellular models, we find that RNAs expressed as cis-NATs tend to co-localize to specific subcellular fractions. This work suggests that post-transcriptional interactions between RNAs with complementary sequences can dictate their localization fate in the cytoplasm.

A ribosomal protein S5 isoform is essential for oogenesis and interacts with distinct RNAs in Drosophila melanogaster.

In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. Here, we demonstrate that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We conclude that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.

Biochemical Fractionation of Time-Resolved Drosophila Embryos Reveals Similar Transcriptomic Alterations in Replication Checkpoint and Histone mRNA Processing Mutants.

In higher eukaryotes, maternally provided gene products drive the initial stages of embryogenesis until the zygotic transcriptional program takes over, a developmental process called the midblastula transition (MBT). In addition to zygotic genome activation, the MBT involves alterations in cell-cycle length and the implementation of DNA damage/replication checkpoints that serve to monitor genome integrity. Previous work has shown that mutations affecting histone mRNA metabolism or DNA replication checkpoint factors severely impact developmental progression through the MBT, prompting us to characterize and contrast the transcriptomic impact of these genetic perturbations. In this study, we define gene expression profiles that mark early embryogenesis in Drosophila through transcriptomic analyses of developmentally staged (early syncytial versus late blastoderm) and biochemically fractionated (nuclear versus cytoplasmic) wild-type (wt) embryos. We then compare the transcriptomic profiles of loss-of-function mutants of the Chk1/Grapes replication checkpoint kinase and the stem loop binding protein (SLBP), a key regulator of replication-dependent histone mRNAs. Our analysis of RNA spatial and temporal distribution during embryogenesis offers new insights into the dynamics of early embryogenesis. In addition, we find that grp and Slbp mutant embryos display profound and highly similar defects in gene expression, most strikingly in zygotic gene expression, compromising the transition from a maternal to a zygotic regulation of development.

Data for the generation of RNA spatiotemporal distributions and interpretation of Chk1 and SLBP protein depletion phenotypes during Drosophila embryogenesis.

The data presented in this article is related to the research article entitled "Biochemical Fractionation of Time-Resolved Drosophila Embryos Reveals Similar Transcriptomic Alterations in Replication Checkpoint and Histone mRNA Processing Mutants" (Lefebvre et al., 2017) [1]. This article provides a spatiotemporal transcriptomic analysis of early embryogenesis and shows that mutations in the checkpoint factor grapes/Chk1 and the histone mRNA processing factor SLBP selectively impair zygotic gene expression. Here, lists of transcripts enriched in early syncytial embryos, late blastoderm embryos, cytoplasmic and nuclear extracts of blastoderm embryos are made public, along with transcription factor motif occurrence for genes enriched in each context. In addition, extensive lists of genes down-regulated upon Chk1 and SLBP protein depletion in embryos are released to enable further analyses.

Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development.

The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3'UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila.

ALK-mediated post-transcriptional regulation: focus on RNA-binding proteins.

Extensive research has been carried out in the past two decades to provide insights into the molecular mechanisms by which the Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK) exerts its oncogenic effects. These studies led to the concept that NPM-ALK acts at the transcriptional level through the activation of several transcription factors downstream of many different signaling pathways including JAK3/STAT3, PI3K/AKT and RAS/ERK. Nevertheless, the discovery of several RNA-binding proteins (RBPs) within ALK interactome suggested an additional and complementary role of this oncogenic kinase at the post-transcriptional level. This review gives emerging views in ALK-mediated post-transcriptional regulation with a focus on RBPs that are associated with ALK. We will summarize the capacity of NPM-ALK in modulating the biological properties of RBPs and then discuss the role of cytoplasmic aggregates, called AGs for "ALK granules", which are observed in anaplastic large cell lymphoma (ALCL) expressing the ALK kinase. AGs contain polyadenylated mRNAs and numerous RBPs but are distinct from processing bodies (PBs) and stress granules (SGs), two well-known discrete cytoplasmic sites involved in mRNA fate.

Developmentally regulated elimination of damaged nuclei involves a Chk2-dependent mechanism of mRNA nuclear retention.

The faithful execution of embryogenesis relies on the ability of organisms to respond to genotoxic stress and to eliminate defective cells that could otherwise compromise viability. In syncytial-stage Drosophila embryos, nuclei with excessive DNA damage undergo programmed elimination through an as-yet poorly understood process of nuclear fallout at the midblastula transition. We show that this involves a Chk2-dependent mechanism of mRNA nuclear retention that is induced by DNA damage and prevents the translation of specific zygotic mRNAs encoding key mitotic, cytoskeletal, and nuclear proteins required to maintain nuclear viability. For histone messages, we show that nuclear retention involves Chk2-mediated inactivation of the Drosophila stem loop binding protein (SLBP), the levels of which are specifically depleted in damaged nuclei following Chk2 phosphorylation, an event that contributes to nuclear fallout. These results reveal a layer of regulation within the DNA damage surveillance systems that safeguard genome integrity in eukaryotes.

Whole mount RNA fluorescent in situ hybridization of Drosophila embryos.

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

HuR-mediated control of C/EBPbeta mRNA stability and translation in ALK-positive anaplastic large cell lymphomas.

The CCAAT/enhancer-binding protein ß (C/EBPß) plays a major role in the pathogenesis of anaplastic large cell lymphomas (ALCL) that express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) tyrosine kinase (ALK(+)). Although ALK-mediated C/EBPß transcriptional activation has been reported, C/EBPß mRNA possesses U- and AU-rich domains in its 3'-untranslated region (3'-UTR) that might be privileged targets for posttranscriptional control in ALK(+) ALCLs. The purpose of this study was to explore this possibility. By using human ALCL-derived cells and a murine model of ALK-transformed cells, we show that the AU-binding protein HuR binds to the 3'-UTR of C/EBPß mRNA, as previously reported in adipocytes, and that NPM-ALK enhances this interaction. Interaction between HuR and C/EBPß mRNA impacts on C/EBPß gene expression at both the mRNA and protein levels. Indeed, C/EBPß mRNA stability following HuR silencing is reduced and reaches the value observed in ALK-inactivated cells. Remarkably, HuR expression is not modified by NPM-ALK, but its association with actively translating polysomes is dramatically increased in ALK(+) cells. HuR/polysomes association diminishes when NPM-ALK activity is inhibited and is accompanied by a concomitant decrease of C/EBPß mRNA translation. Finally, we show that HuR and NPM-ALK colocalized in cytoplasmic granules and HuR is phosphroylated on tyrosine residues in ALK(+) ALCL cells. Our study thus demonstrates that C/EBPß is indeed regulated at the posttranscriptional level by HuR in ALK(+) cells, leading us to propose that part of NPM-ALK oncogenic properties relies on its ability to modify HuR properties in the cytoplasm and hence to alter expression of key actors of transformation.

Oncologic trogocytosis of an original stromal cells induces chemoresistance of ovarian tumours.

BACKGROUND: The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours. METHODOLOGY/PRINCIPAL FINDINGS: We isolated an original type of stromal cells, referred to as "Hospicells" from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance. CONCLUSIONS/SIGNIFICANCE: This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient's tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.