The fate of RNA in Cyanobacteria: subcellular transcriptome organizationin Synechocystis sp. PCC 6803

Eukaryotic and bacterial cells achieve spatial organization by partitioning biochemical processes across subcellular areas. Beyond protein and lipid sorting, bacterial RNA localization is emerging as an organizing principle of spatial gene expression. In E. coli, mRNAs, tRNAs, rRNAs, and regulatory RNAs occupy distinct subcellular positions. In this study, we established RNA localization sequencing (Rloc-seq) in Synechocystis to map RNA localization between combined membrane fraction (thylakoid and plasma membrane) and the cytosol. We then validated individual membrane- and cytosol-enriched transcripts at single cell resolution by RNA-Fluorescence in situ hybridization. Our data show that the Synechocystis sp. PCC 6803 transcriptome is spatially organized: for example, mRNAs encoding photosystem I/II components and ribosomal proteins are significantly enriched in the membrane fraction and localize at the inner thylakoid membrane surface, matching the destination of their encoded proteins. 16S rRNA FISH highlights ribosome-rich zones proximal to the thylakoid membranes, supporting membrane-proximal translation. Inhibiting translation with puromycin alters the abundance of a subset of RNAs but leaves many localization patterns intact, indicating that a substantial fraction of RNA positioning occurs via translation-independent mechanisms. To uncover the mechanisms underlying differential transcriptome organization, we characterized the cyanobacterial RRM-motif-containing RNA-binding protein Rbp3. Rbp3 binds RNA in vivo, associates with ribosomal proteins, and binds uracil-rich motifs within the 3’UTR or coding regions of target transcripts. These targets are overrepresented among membrane-enriched RNAs, even when translation is inhibited, supporting RBP-mediated transport of specific mRNAs to their subcellular destinations. Complementary work in E. coli has demonstrated that intrinsic mRNA features can drive membrane targeting: uracil-rich, sequential, and structural motifs promote association with the inner membrane. This mechanism can act as an alternative to SRP-dependent protein targeting when the pathway is impaired, with SecYEG and YidC serving as RNA docking sites. Finally, we analyzed the 5’-sensing function of Synechocystis RNase E by generating a loss-of-function mutant strain and mapping the transcriptome-wide cleavage sites. Altogether, our data support a model in which spatial RNA positioning acts upstream of, or in parallel with, co-translational membrane insertion. We propose that RNA-encoded features, recognized by specific RNA-binding proteins, determine subcellular localization and may sort transcripts to the appropriate membrane system in Synechocystis