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.

Flying the RNA Nest: Drosophila Reveals Novel Insights into the Transcriptome Dynamics of Early Development.

Early development is punctuated by a series of pervasive and fast paced transitions. These events reshape a differentiated oocyte into a totipotent embryo and allow it to gradually mount a genetic program of its own, thereby framing a new organism. Specifically, developmental transitions that ensure the maternal to embryonic control of developmental events entail a deep remodeling of transcriptional and transcriptomic landscapes. Drosophila provides an elegant and genetically tractable system to investigate these conserved changes at a dazzling developmental pace. Here, we review recent studies applying emerging technologies such as ribosome profiling, in situ Hi-C chromatin probing and live embryo RNA imaging to investigate the transcriptional dynamics at play during Drosophila embryogenesis. In light of this new literature, we revisit the main models of zygotic genome activation (ZGA). We also review the contributions played by zygotic transcription in shaping embryogenesis and explore emerging concepts of processes such as transcriptional bursting and transcriptional memory.

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.

Comparative transcriptomic analysis of human and Drosophila extracellular vesicles.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that human and Drosophila cells release similar EVs with diameters ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNAs, related pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these sizeable similarities suggest that EVs could potentially enable RNA-mediated intercellular communication in Drosophila.

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.