Grad-seq analysis of a photosynthetic bacterium rich in internal membrane systems

Regulatory sRNAs in photosynthetic cyanobacteria have been reported, but the lack of plausible RNA chaperones involved in this regulation appears enigmatic. Here, we analyzed the full ensemble of cellular RNAs and cofractionating proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803, a cyanobacterium rich in internal membrane systems. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished based on the in-gradient protein distribution supporting the validity of the approach. We determined the sedimentation profiles of ~67.3% of all annotated proteins and ~62.2% of all transcripts. A clustering approach of the in-gradient distribution profiles supported the mutually exclusive association of either RpoZ or the 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes. Focusing on ribonucleoprotein complexes, the data suggest the existence of previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. The resulting short list of potential RNA chaperones contains a YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The dataset is available at https://trex90.shinyapps.io/GradSeqExplorer_SynPCC6803/, providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.