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Dynamic Mapping of RNA-Binding Proteins During Bacillus subtilis Sporulation Using Orthogonal Organic Phase Separation
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DOI：10.21769/BioProtoc.5622.png)
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Abstract
En 中文
RNA-binding proteins (RBPs) have pleiotropic roles in modulating the physiology of both eukaryotic and prokaryotic cells, enabling them to adapt to environmental variations. The importance of RBPs has led to the development of a variety of methods aiming to identify them. However, most of these approaches have primarily been implemented and optimized in eukaryotic systems. To both uncover novel RBPs involved in Bacillus subtilis sporulation and capture their RNA-binding ability dynamically, we adapted the orthogonal organic phase separation technique (OOPS), which had previously been used in Escherichia coli to reveal its RNA-binding proteome (RBPome). We optimized the UV cross-linking process used to stabilize RNA-protein interactions in vivo and the bacterial lysis process to overcome the robust cell wall of Gram-positive sporulating cells. RNA-protein complexes are then recovered after phase separation steps using guanidinium thiocyanate- phenol-chloroform, and RNA-associated proteins are identified and label-free-quantified by liquid chromatography-mass spectrometry. Collecting samples at various time points during sporulation further enables tracking the dynamics of the RBPome. In addition to being applicable to bacteria and requiring minimal starting material, this method has provided a comprehensive map of the RBPome during sporulation, refining the roles of known factors and revealing new players.
Keywords:
RNA binding proteins
RBPome
Bacillus subtilis
OOPS
UV cross-linking
Adaptation
Proteomics
Sporulation
Journal
B
IF:
1.1
Papers:
191
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5.4K
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