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

BACKGROUND: Autogenous 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. RESULTS: We 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. CONCLUSIONS: This 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.

Bacterial RNA motif in the 5' UTR of rpsF interacts with an S6:S18 complex.

Approximately half the transcripts encoding ribosomal proteins in Escherichia coli include a structured RNA motif that interacts with a specific ribosomal protein to inhibit gene expression, thus allowing stoichiometric production of ribosome components. However, many of these RNA structures are not widely distributed across bacterial phyla. It is increasingly common for RNA motifs associated with ribosomal protein genes to be identified using comparative genomic methods, yet these are rarely experimentally validated. In this work, we characterize one such motif that precedes operons containing rpsF and rpsR, which encode ribosomal proteins S6 and S18. This RNA structure is widely distributed across many phyla of bacteria despite differences within the downstream operon, and examples are present in both E. coli and Bacillus subtilis. We demonstrate a direct interaction between an example of the RNA from B. subtilis and an S6:S18 complex using in vitro binding assays, verify our predicted secondary structure, and identify a putative protein-binding site. The proposed binding site bears a strong resemblance to the S18 binding site within the 16S rRNA, suggesting molecular mimicry. This interaction is a valuable addition to the canon of ribosomal protein mRNA interactions. This work shows how experimental verification translates computational results into concrete knowledge of biological systems.

RNA structures regulating ribosomal protein biosynthesis in bacilli.

In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species.

Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria.

In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria.

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.