R-DeeP/TripepSVM identifies the RNA-binding OB-fold-like protein PatR as regulator of heterocyst patterning

Abstract

RNA-binding proteins (RBPs) are central components of gene regulatory networks. The differentiation of heterocysts in filamentous cyanobacteria is an example of cell differentiation in prokaryotes. Although multiple non-coding transcripts are involved in this process, no RBPs have been implicated thus far. Here we used quantitative mass spectrometry to analyze the differential fractionation of RNA–protein complexes after RNase treatment in density gradients yielding 333 RNA-associated proteins, while a bioinformatic prediction yielded 311 RBP candidates in Nostoc sp. PCC 7120. We validated in vivo the RNA-binding capacity of six RBP candidates. Some participate in essential physiological aspects, such as photosynthesis (Alr2890), thylakoid biogenesis (Vipp1) or heterocyst differentiation (PrpA, PatU3), but their association with RNA was unknown. Validated RBPs Asl3888 and Alr1700 were not previously characterized. Alr1700 is an RBP with two oligonucleotide/oligosaccharide-binding (OB)-fold-like domains that is differentially expressed in heterocysts and interacts with non-coding regulatory RNAs. Deletion of alr1700 led to complete deregulation of the cell differentiation process, a striking increase in the number of heterocyst-like cells, and was ultimately lethal in the absence of combined nitrogen. These observations characterize this RBP as a master regulator of the heterocyst patterning and differentiation process, leading us to rename Alr1700 to PatR.