Biology subjects

Yassine, H.

Publications and source records attributed to Yassine, H..

3 recordsLinked to original sources

bioRxiv2024

Stress Changes the Bacterial Biomolecular Condensate Material State and Shifts Function from mRNA Decay to Storage

Bacterial ribonucleoprotein bodies (BR-bodies) are dynamic biomolecular condensates that play a pivotal role in RNA metabolism. We investigated how BR-bodies significantly influence mRNA fate by transitioning between liquid- and solid-like states in response to stress. With a combination of single-molecule and bulk fluorescence microscopy, biochemical assays, and quantitative analyses, we determine that BR-bodies promote efficient mRNA decay in a liquid-like condensate during exponential growth. On the other hand, BR-bodies are repurposed from sites of mRNA decay to reservoirs for mRNA storage under stress; a functional change that is enabled by their transition to a more rigid state, marked by reduced internal dynamics, increased molecular density, and prolonged residence time of ribonuclease E. Furthermore, we manipulated ATP levels and translation rates, and we conclude that the accumulation of ribosome-depleted mRNA is a key factor driving BR-body rigification, and that condensate maturation further contributes to this process. Upon nutrient replenishment, stationary-phase BR-bodies disassemble, releasing stored mRNAs for rapid translation, demonstrating that BR-body function is governed by a reversible mechanism for resource management. These findings reveal adaptive strategies by which bacteria regulate RNA metabolism through condensate-mediated control of mRNA decay and storage.

biophysics↗

bioRxiv2024

APEX2 proximity labeling of RNA in bacteria

Rapid spatially controlled methods are needed to investigate RNA localization in bacterial cells. APEX2 proximity labeling was shown to be adaptable to rapid RNA labeling in eukaryotic cells, and through the fusion of APEX2 to different proteins targeted to different subcellular locations, has been useful to identify RNA localization in these cells. Therefore, we adapted APEX2 proximity labeling of RNA to bacterial cells by generating an APEX2 fusion to the RNase E gene, which is necessary and sufficient for BR-body formation. APEX2 fusion is minimally perturbative and RNA can be rapidly labeled on the sub-minute timescale with Alkyne-Phenol, outpacing the rapid speed of mRNA decay in bacteria. Alkyne-Phenol provides flexibility in the overall downstream application with copper catalyzed click-chemistry for downstream applications, such as fluorescent dye-azides or biotin-azides for purification. Altogether, APEX2 proximity labeling of RNA provides a useful method for studying RNA localization in bacteria. MotivationStudies over the past several years have shown that distinct RNAs can be targeted to subcellular locations in bacterial cells. The ability to investigate localized RNAs in bacteria is currently limited to imaging-based approaches or to laborious procedures to isolate ribonucleoprotein complexes by grad-seq, HITS-CLIP, or Rloc-seq. However, a major challenge in studying mRNA localization in bacterial cells is that bacterial mRNAs typically last for only a few minutes in the cell, while experiments to investigate their localization or interaction partners can take much longer. Therefore, rapid methods of studying RNA localization are needed to bridge this technical challenge. HighlightsO_LIAPEX2 proximity labeling can be applied to RNA in bacteria C_LIO_LIAPEX2 RNA labeling reactions occur on the sub-minute timescale. C_LIO_LIAPEX2 workflow requires less material and time than current methods. C_LIO_LIAlkyne-Phenol APEX2 substrate provides flexibility with click-chemistry. C_LI

microbiology↗

bioRxiv2023

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

Bacterial RNP bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We found 111 BR-body enriched proteins, including several RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, showing BR-bodies are more complex than previously assumed. While most BR-body enriched proteins that were tested cannot phase separate, we identified five that undergo RNA-dependent phase separation in vitro, showing other RNP condensates interface with BR-bodies. RNA degradosome protein clients are recruited more strongly to RNase E droplets than droplets of other RNP condensates, implying that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble with preferred directionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing. HighlightsO_LIBR-body proteomics identified 111 proteins enriched in BR-bodies. C_LIO_LIBR-bodies associate with an interconnected network of RNP condensates. C_LIO_LIBR-body condensation is modulated by its interaction network. C_LIO_LIRNA is required for rapid BR-body condensation. C_LI Graphical AbstractSummary of the BR-body protein interactome. Lines between two protein circles represent a direct interaction.

microbiology↗