The RRM domain-containing protein Rbp3 interacts with ribosomes and the 3' ends of mRNAs encoding photosynthesis proteins

Abstract

RNA recognition motif (RRM) domain proteins are crucial RNA-binding proteins (RBPs) across all domains of life. In cyanobacteria, single RRM domain proteins are involved in mRNA targeting to the thylakoid membrane and acclimation to certain stress conditions, but many details of their physiological functions and molecular targets have remained unknown. The model cyanobacterium Synechocystis sp. PCC 6803 has a family of three genes encoding the RRM domain-containing proteins Rbp1, Rbp2 and Rbp3. Here, we verified the RNA-binding activity of Rbp3 in vivo and show that cells of a {Delta}rbp3 deletion strain had a lower PSI:PSII ratio and decreased pigment content and were significantly smaller than wild-type cells. To identify the set of interacting molecules, co-immunoprecipitation experiments were performed with a strain expressing a C-terminally FLAG-tagged Rbp3. Mass spectrometry of the elution fraction suggested physical proximity between Rbp3, ribosomes, and a very small number of other proteins. The most highly enriched transcript in the co-eluting RNA fraction was the psaAB mRNA. This was corroborated by fluorescent in situ hybridization (FISH) analyses showing decreased psaA mRNA signals in {Delta}rbp3, and colocalization with Rbp3-GFP in the wild type. Other mRNAs co-enriched with Rbp3 encode thylakoid, plasma membrane and carboxysome proteins. Binding assays using Bio-layer Interferometry validated the Rbp3-psaAB mRNA interaction, indicating a preference for folded RNA segments near or overlapping the respective stop codons. Significance statementThe mechanisms by which proteins are produced at specific sites and inserted into the intricate membrane systems of photosynthetic cyanobacteria are only partially understood. While RRM domain proteins are well-studied RNA-binding proteins in eukaryotes, their functions in bacteria remain underexplored. This study reveals that the RRM domain protein Rbp3 in the cyanobacterium Synechocystis sp. PCC 6803 binds mRNAs encoding photosynthetic proteins, plasma membrane proteins and carboxysome proteins and localizes near ribosomes. The bound RNA segments are typically near the ends of coding regions, or in 5' untranslated regions. These findings suggest that Rbp3 is involved in targeting mRNAs to various intracellular locations by interacting with structural elements within these mRNA molecules.