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 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 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.
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.
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.
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