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

Abstract

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.

Biological functions, regulatory mechanisms, and disease relevance of RNA localization pathways

“…This type of analysis can thus be useful in identifying molecular machines that are likely enriched at specific subcellular compartments through localized translation. Finally, similar to mRNA profiling efforts, these types of subcellular transcriptomics studies have also revealed the extensive asymmetry in distribution patterns displayed by various families of noncoding RNAs, including lncRNAs, miRNAs, and circular RNAs . Collectively, these approaches highlight that high prevalence and diversity of RNA localization events.…”

“…Using a combined approach of RNA‐seq and MS profiling of human and Drosophila cells fractionated into distinct biochemically defined compartments (i.e., nuclear, insoluble, endomembrane, and cytosol), Benoit Bouvrette et al . found that the majority of mRNAs and noncoding RNA biotypes are asymmetrically distributed in patterns that are generally conserved evolutionarily . Comparative analysis of mRNA and protein distribution profiles revealed a general lack of correlation, a feature that was strongly influenced by specific families of transcripts exhibiting strong asymmetric distribution with their encoded protein products.…”

“…This work showed that hundreds of mRNAs are transported from the basal lamina to the ribosome‐enriched apical cytoplasm of intestinal epithelial cells when fasted mice were refed, thereby revealing the dynamic nature of global cellular mRNA arrangements . Several groups have also simultaneously profiled RNA and protein expression across subcellular compartments in order to assess the general coherence in expression signatures . For instance, Zappulo et al .…”

“…For instance, mRNAs encoding DNA‐binding transcription factors and chromatin components/regulators need to undergo translation in the cytoplasm, while their protein products are typically strongly enriched in the nucleus. However, when this fractionation data was dissected further and segmented to interrogate mRNAs encoding components of known protein modules, functionally‐related transcripts tended to exhibit similar fractionation profiles, while the degree of co‐fraction with their encoded proteins varied depending on protein complexes under study . For example, mRNAs for components of the Arp2‐3 complex, known to undergo transport to cell protrusions , showed close co‐fractionation with the their protein products.…”

Subcellular mRNA localization regulates ribosome biogenesis in migrating cells

“…It is now clear that rather than being uniformly distributed throughout the cytoplasm, the 50 majority of eukaryotic mRNAs exhibit specific subcellular localizations (Benoit Bouvrette et al, 2018;Lecuyer et al, 2007;Wang et al, 2012;Wilk et al, 2016). Such localization can act as a means of localizing the encoded proteins (Zappulo et al, 2017), or function instead as a mechanism for post-transcriptional regulation by modulating the access of mRNAs to different trans-acting factors (Kejiou and Palazzo, 2017;Pizzinga et al, 2019;Prasanth et al, 55 2005).…”