Biology subjects

LeBlanc, H.

Publications and source records attributed to LeBlanc, H..

5 recordsLinked to original sources

bioRxiv2026

RNase J2 is a specificity factor that governs global mRNA stability in Bacillus subtilis

Ribonucleases (RNases) play a key role in modulating gene expression at the post-transcriptional level, enabling bacteria to rapidly adapt to their environment. The B. subtilis genome encodes more than 20 RNases and understanding the role of each of these enzymes is crucial for a comprehensive knowledge of bacterial post-transcriptional adaptation mechanisms. Among these, RNase J1 and J2, belonging to the {beta}-lactamase enzyme family, form a heterodimer. While the primary function of RNase J1 as a 5'-exoribonuclease in B. subtilis has been known for some time, the role of RNase J2, encoded by the rnjB gene, has remained unclear. Indeed, it has been considered a minor degradation factor due its very weak 5'-exoribonuclease in vitro, the lack of a growth phenotype and the limited number of mRNAs with altered equilibrium levels in rnjB mutants. In this study, we demonstrate that the absence of RNase J2 influences pellicle and macro-colony biofilm formation, as well as swarming in B. subtilis. We show that RNase J2 plays a far more global role in governing mRNA half-lives than originally thought, by stimulating RNase J1-mediated degradation of a subset of RNase J1's mRNA substrates. An inter-subunit contact between RNase J1 and J2 is crucial for RNase J2 action. Altogether, our results suggest that RNase J2 acts primarily as a specificity co-factor for RNase J1 rather than as a ribonuclease per se.

microbiology↗

bioRxiv2024

Improved constructs for bait RNA display in a bacterial three-hybrid assay

We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.

molecular biology↗

bioRxiv2022

Chronic Gq activation of ventral hippocampal neurons and astrocytes differentially affects memory and behavior

Network dysfunction is implicated in numerous diseases and psychiatric disorders, and the hippocampus serves as a common origin for these abnormalities. To test the hypothesis that chronic modulation of neurons and astrocytes induces impairments in cognition, we activated the hM3D(Gq) pathway in CaMKII+ neurons or GFAP+ astrocytes within the ventral hippocampus across 3, 6 and 9 months. CaMKII-hM3Dq activation impaired fear extinction at 3 months and acquisition at 9 months. Both CaMKII-hM3Dq manipulation and aging had differential effects on anxiety and social interaction. GFAP-hM3Dq activation impacted fear memory at 6 and 9 months. GFAP-hM3Dq activation impacted anxiety in the open field only at the earliest time point. CaMKII-hM3Dq activation modified the number of microglia, while GFAP-hM3Dq activation impacted microglial morphological characteristics, but neither affected these measures in astrocytes. Overall, our study elucidates how distinct cell types can modify behavior through network dysfunction, while adding a more direct role for glia in modulating behavior. HighlightsO_LICaMKII- and GFAP-Gq activation impacted memory, anxiety, and social behaviors. C_LIO_LINovel environment exploration was affected by CaMKII- and GFAP-Gq activation. C_LIO_LICaMKII-Gq modified microglial number, while GFAP-Gq affected microglial morphology. C_LIO_LINeither cell manipulation affected astrocytic number or morphology. C_LI

animal behavior and cognition↗

bioRxiv2020

Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter-protein

Non-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system, and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system, and will facilitate exploration of the binding mechanisms of these interactions.

microbiology↗

bioRxiv2019

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex.

biochemistry↗