Discovery and validation of novel and distinct RNA regulators for ribosomal protein S15 in diverse bacterial phyla

Abstract

BackgroundAutogenous cis-regulators of ribosomal protein synthesis play a critical role in maintaining the stoichiometry of ribosome components. Structured portions within an mRNA transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units. Three distinct RNA structures from different bacterial phyla have demonstrated interactions with S15 to regulate gene expression; however, these RNAs are distributed across a small fraction of bacterial diversity.ResultsWe used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative RNA structures associated with the S15 coding region in microbial genomes. These structures are completely distinct from those previously published and encompass potential regulatory regions including ribosome-binding sites. To validate the biological relevance of our findings, we demonstrate that an example of the Alphaproteobacterial RNA from Rhizobium radiobacter specifically interacts with S15 in vitro, and allows in vivo regulation of gene expression in an E. coli reporter system. In addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction.ConclusionsThis work illustrates the importance of integrating comparative genomic and transcriptomic approaches during de novo ncRNA identification and reveals a diversity of distinct natural RNA regulators that support analogous biological functions. Furthermore, this work indicates that many additional uncharacterized RNA regulators likely exist within bacterial genomes and that the plasticity of RNA structure allows unique, and likely independently derived, solutions to the same biological problem.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-657) contains supplementary material, which is available to authorized users.

An S6:S18 complex inhibits translation of E. coli rpsF

“…Upon expression of the first three genes of the rpsF operon (rpsF-priB-rpsR), we observe a large decrease in β-galactosidase activity (approximately fourfold repression). To control for potential global changes in translational efficiency that may be due to ribosome defects associated with overexpression of S6 and S18, we also examined the β-galactosidase activity of a similar lacZ fusion with an RNA structure not expected to interact with S6 or S18, that preceded ribosomal protein S15 in E. coli (rpsO ′ -′ lacZ) (Philippe et al 1993;Slinger et al 2014). Although the rpsO ′ -′ lacZ fusion results in higher β-galactosidase activity in comparison to the rpsF_leader-lacZ constructs (Fig.…”

“…To assess the regulatory ability of the mRNA sequence preceding the rpsF gene in E. coli (rpsF_leader), we constructed a translational fusion between the RNA structure, including the first nine codons of rpsF, and lacZ, under transcriptional control of the Lac promoter (Slinger et al 2014). To supply potential exogenous protein regulatory partners, we amplified portions of the rpsF operon and overexpressed them under the control of an arabinose inducible promoter on pBAD33 (Fig.…”

“…The lacZ reporter plasmid is a modified version of the pLacThiM#2-tetA-gfpuv plasmid previously described (Muranaka et al 2009;Slinger et al 2014), in which the tetA-gfpuv construct is replaced with a lacZ gene using SalI and XbaI restriction sites. The fragment corresponding to the RNA and the first nine codons of rpsF (NC_000913.3/4425023-4425144) was PCR amplified from genomic DNA extracted from E. coli strain XL1-Blue (Agilent Technologies) and cloned between the EcoRI and SalI sites, to generate an RNA-lacZ translational fusion.…”

“…Instead it appears that alternative RNA regulators with distinct structures and mechanisms of action often accomplish analogous functions in different phyla of bacteria (Guillier et al 2002;Choonee et al 2007;Slinger et al 2014). The diversity of RNA structures regulating ribosomal proteins follows the general trend that RNA regulators in bacteria are typically not widely conserved but evolve rapidly (Peer and Margalit 2011) and are present only in relatively narrow groups of bacteria (Lindgreen et al 2014).…”

Fitness advantages conferred by the L20-interacting RNAcis-regulator of ribosomal protein synthesis inBacillus subtilis

“…Nearly all bacterial phyla possess at least one of the widely distributed cis-regulatory RNA structures (Fu et al 2013), setting the precedent for RNA-mediated autogenous regulation of ribosomal protein synthesis throughout bacteria. Furthermore, RNA structures that perform analogous regulatory functions in response to homologous proteins, yet share little to no similarity to the regulatory RNAs in E. coli, have been identified in different bacterial phyla (Grundy and Henkin 1991;1992;Scott and Williamson 2001;Serganov et al 2003;Choonee et al 2007;Deiorio-Haggar et al 2013;Slinger et al 2014). These distinct regulatory RNA structures are typically narrowly distributed to specific bacterial groups and are likely the result of multiple instances of independent evolution.…”