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 cyanobacteriumSynechocystissp. 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 Rbp3in vivoand show that cells of a Δrbp3deletion strain had a lower PSI:PSII ratio and 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 thepsaABmRNA. This was corroborated by fluorescentin situhybridization (FISH) analyses showing decreasedpsaAmRNA signals and colocalization with Rbp3-GFP signals and ribosomes. Other enriched mRNAs encode thylakoid, plasma membrane and carboxysome proteins. The Rbp3-mRNA interactions occurred preferentially towards the end of coding regions or the 3′UTRs, although some were also mapped to other regions. Binding assays using Bio-layer Interferometry validated the Rbp3-psaABmRNA 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 cyanobacteriumSynechocystissp. 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 specific intracellular locations by interacting with structural elements within these mRNA molecules.