Understanding how bacteria cause disease requires knowledge of which genes are expressed and how they are regulated during infection. While RNA-seq is now a routine method for gene expression analysis in bacterial pathogens, the past years have also witnessed a surge of novel RNA-seq based approaches going beyond standard mRNA profiling. These include variations of the technique to capture post-transcriptional networks controlled by small RNAs and to discover associated RNA-binding proteins in the pathogen itself. Dual RNA-seq analyzing pathogen and host simultaneously has revealed roles of noncoding RNAs during infection and enabled the correlation of bacterial gene activity with specific host responses. Single-cell RNA-seq studies have addressed how heterogeneity among individual host cells may determine infection outcomes.
- A surge of new RNA-seq based methods to study bacterial pathogenesis.
- RIP-seq, CLIP-seq, CLASH, RIL-seq, GRIL-seq, MAPS and Term-seq discover new RNA-based regulations.
- Grad-seq guides the discovery of RNA-binding proteins.
- Dual RNA-seq for simultaneous transcriptomics in host–pathogen interactions.
- Single-cell RNA-seq addresses heterogeneity in infections.
New RNA-seq based methods to decipher regulatory RNA networks
MAPS (MS2-affinity purification coupled with RNA-seq): an MS2 affinity tag is fused to a sRNA of interest and co-purified with its interacting RNA and sequenced. GRIL-seq (Global sRNA target identification by ligation and sequencing): the method allows the identification of direct targets of sRNAs by in vivo proximity ligation. It is similar to MAPS, but additionally ligates short sRNA–mRNA duplexes in vivo by co-expressing T4 RNA ligase before capturing candidate target mRNAs with a sRNA-specific oligonucleotide. RIP-seq (native RNA immunoprecipitation followed by RNA-seq): target transcripts are immunoprecipated with a protein of interest in cell lysates and analysed by RNA-seq. CLIP-seq (cross-linking immunoprecipitation-high-throughput sequencing): in vivo UV treatment covalently cross-links RNA to proteins before co-purification and RNA-seq. RNA trimming by ribonucleases outside of the binding region enables to map the binding region at single-nucleotide resolution. CLASH (crosslinking, ligation, and sequencing of hybrids): RNA molecules are UV-crosslinked to a flagged RBP in vivo. Next, the bound RNA is trimmed using an RNase and RNA linkers are ligated to the immobilized RNA molecules in the RNA–RBP complexes. Finally, coupled RNA molecules are ligated into one single molecule of two different types, either single or chimeric fragments. RIL-seq (RNA interaction by ligation and sequencing): a protein-RNA complex is immunoprecipitated and bound RNAs are crosslinked to the protein. After enzymatic digestion, RNAs are ligated and subjected to RNA-seq. RNA–RNA interactions are revealed after mapping and identification of chimeric reads.