Biology subjects

Vanderpool, C. K.

Publications and source records attributed to Vanderpool, C. K..

4 recordsLinked to original sources

bioRxiv2018

sRNA-target Prediction Organizing Tool (SPOT) integrates computational and experimental data to facilitate functional characterization of bacterial small RNAs

Small RNAs (sRNAs) post-transcriptionally regulate mRNA targets, typically under conditions of environmental stress. Although hundreds of sRNAs have been discovered in diverse bacterial genomes, most sRNAs remain uncharacterized, even in model organisms. Identification of mRNA targets directly regulated by sRNAs is rate-limiting for sRNA functional characterization. To address this, we developed a computational pipeline that we named SPOT for sRNA-target Prediction Organizing Tool. SPOT incorporates existing computational tools to search for sRNA binding sites, allows filtering based on experimental data, and organizes the results into a standardized report. SPOT sensitivity (Correctly Predicted Targets/Total Known Targets) was equal to or exceeded any individual method when used on 12 characterized sRNAs. Using SPOT, we generated a set of target predictions for the sRNA RydC, which was previously shown to positively regulate cfa mRNA, encoding cyclopropane fatty acid synthase. SPOT identified cfa along with additional putative mRNA targets, which we then tested experimentally. Our results demonstrated that in addition to cfa mRNA, RydC also regulates trpE and pheA mRNAs, which encode aromatic amino acid biosynthesis enzymes. Our results suggest that SPOT can facilitate elucidation of sRNA target regulons to expand our understanding of the many regulatory roles played by bacterial sRNAs.\n\nIMPORTANCESmall RNAs (sRNAs) regulate gene expression in diverse bacteria by interacting with mRNAs to change their structure, stability or translation. Hundreds of sRNAs have been identified in bacteria, but characterization of their regulatory functions is limited by difficulty with sensitive and accurate identification of mRNA targets. Thus, new robust methods of bacterial sRNA target identification are in demand. Here, we describe our Small RNA-target Prediction Organizing Tool, which streamlines the process of sRNA target prediction by providing a single pipeline that combines available computational prediction tools with customizable results filtering based on experimental data. SPOT allows the user to rapidly produce a prioritized list of predicted sRNA-target mRNA interactions that serves as a basis for further experimental characterization. This tool will facilitate elucidation of sRNA regulons in bacteria, allowing new discoveries regarding the roles of sRNAs in bacterial stress responses and metabolic regulation.

microbiology↗

bioRxiv2018

Multiple Small RNAs Alter Membrane Lipid Composition via Post-Transcriptional Regulation of Cyclopropane Fatty Acid Synthase

Altering membrane protein and lipid composition is an important strategy for maintaining membrane integrity during environmental stress. Many bacterial small RNAs (sRNAs) control membrane protein production, but sRNA-mediated regulation of membrane fatty acid composition is less understood. The sRNA RydC was previously shown to stabilize cfa (cyclopropane fatty acid synthase) mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. Here, we report that three additional sRNAs, ArrS, CpxQ, and GadF also regulate cfa post-transcriptionally. RydC, ArrS, and GadF act through masking an RNase E cleavage site in the cfa mRNA 5 untranslated region (UTR), and all three sRNAs post-transcriptionally activate cfa. In contrast, CpxQ binds to a different site in the cfa mRNA 5 UTR and represses cfa. Alteration of membrane lipid composition is a key mechanism for bacteria to survive low pH environments, and we show that cfa translation increases in an sRNA-dependent manner when cells are subjected to mild acid stress. This work suggests an important role for sRNAs in the acid stress response through regulation of cfa mRNA.

microbiology↗

bioRxiv2017

Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

The mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.\n\nIMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of \"one sRNA-one target\", and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.

microbiology↗

bioRxiv2017

Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism

In bacteria, the canonical mechanism of translational repression by small RNAs (sRNAs) involves sRNA-mRNA base pairing that occludes the ribosome binding site (RBS), directly preventing translation. In this mechanism, the sRNA is the direct regulator, while the RNA chaperone Hfq plays a supporting role by stabilizing the sRNA. There are a few examples where the sRNA does not directly interfere with ribosome binding, yet translation of the target mRNA is still inhibited. Mechanistically, this non-canonical regulation by sRNAs is poorly understood. Our previous work demonstrated repression of the mannose transporter manX mRNA by the sRNA SgrS, but the regulatory mechanism was unknown. Here, we report that manX translation is controlled by a molecular role-reversal mechanism where Hfq, not the sRNA, is the direct repressor. Hfq binding adjacent to the manX RBS is required for sRNA-mediated translational repression. Translation of manX is also regulated by another sRNA, DicF, via the same non-canonical Hfq-dependent mechanism. Our results suggest that the sRNAs recruit Hfq to its binding site or stabilize the mRNA-Hfq complex. This work adds to the growing number of examples of diverse mechanisms of translational regulation by sRNAs in bacteria.

microbiology↗