Defining Bacterial RNA-RNA Interactomes Using CLASH.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen accounting for high mortality rates among infected patients. Transcriptomic regulation by small RNAs (sRNAs) has been shown to regulate networks promoting antibiotic resistance and virulence in S. aureus. Yet, the biological role of most sRNAs during MRSA host infection remains unknown. To fill this gap, in collaboration with the lab of Jai Tree, we performed comprehensive RNA-RNA interactome analyses in MRSA using CLASH under conditions that mimic the host environment. Here we present a detailed version of this optimized CLASH (cross-linking, ligation, and sequencing of hybrids) protocol we recently developed, which has been tailored to explore the RNA interactome in S. aureus as well as other Gram-positive bacteria. Alongside, we introduce a compilation of helpful Python functions for analyzing folding energies of putative RNA-RNA interactions and streamlining sRNA and mRNA seed discovery in CLASH data. In the accompanying computational demonstration, we aim to establish a standardized strategy to evaluate the likelihood that observed chimeras arise from true RNA-RNA interactions.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins.

Advantages and limitations of UV cross-linking analysis of protein-RNA interactomes in microbes.

RNA-binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein-RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. The recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross-linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV-dependent techniques to inspect RNA-binding activity.

Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system.

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli. Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice.