LIGR-Nue for the global mapping of the bacterial sRNA targetome in vivo

Interactions between regulatory small RNAs (sRNAs) and their target transcripts are central for post-transcriptional regulation in bacteria. Based on RNA-seq analysis, we determined the relative abundance of several sRNAs in the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 under photoautotrophic growth conditions and after transfer to darkness. We observed an increase in the abundance of the sRNAs DsiR1 to 58.6% and of CsiR1 to 19% of the sRNA transcriptome after transfer to darkness. We then developed a duplex-directed, transcriptome-wide mapping strategy of RNA-RNA interactions in vivo, called LIGR-Nue, which involves aminomethyltrioxsalen-mediated UV cross-linking, proximity ligation, crosslink reversal, reverse transcription and sequencing. The data were analysed using the RNANUE pipeline for the detection of chimeric reads and mapping, and benchmarked against known targetome data for the sRNAs PsrR1 and NsiR4. Using this methodology, we identified RNA-RNA interactions, such as PsrR1 as a regulator of carbon assimilation, and DsiR1 as a global silencing factor of photosynthesis-associated mRNAs in the dark. This data enables the comprehensive analysis of sRNA functions in post-transcriptional gene regulation. LIGR-Nue is broadly applicable to map RNA-RNA interactions in bacteria.