Biology subjects

Baudet, M.-L.

Publications and source records attributed to Baudet, M.-L..

4 recordsLinked to original sources

bioRxiv2023

Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We now develop a method based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Using the highly conserved Xenopus miR-181a family as a proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting pre-miRNA apical region elicit a precursor-selective inhibition of mature miRNA-5p production. The levels of miRNAs released from the 3'-arm of these precursors are not reduced, suggesting that our approach is also arm-selective. Overall, we show that this strategy can be successfully applied in vivo to achieve high target selectivity to study identical or highly similar miRNAs stemming from different precursors.

molecular biology↗

bioRxiv2021

SHOT-R: A next generation algorithm for particle kinematics analysis

Analysis of live-imaging experiments is crucial to decipher a plethora of cellular mechanisms within physiological and pathological contexts. Kymograph, i.e. graphical representations of particle spatial position over time, and single particle tracking (SPT) are the currently available tools to extract information on particle transport and velocity. However, the spatiotemporal approximation applied in particle trajectory reconstruction with those methods intrinsically prevents an accurate analysis of particle kinematics and of instantaneous behaviours. Here, we present SHOT-R, a novel numerical method based on polynomial reconstruction of 4D (3D+time) particle trajectories. SHOT-R, contrary to other tools, computes bona fide instantaneous and directional velocity, and acceleration. Thanks to its high order continuous reconstruction it allows, for the first time, kinematics analysis of co-trafficked particles. Overall, SHOT-R is a novel, versatile, and physically reliable numerical method that achieves all-encompassing particle kinematics studies at unprecedented accuracy on any live-imaging experiment where the spatiotemporal coordinates can be retrieved.

cell biology↗

bioRxiv2021

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is important also for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current methods fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. Additionally, we demonstrated the protocol to be effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids.

cell biology↗

bioRxiv2018

Precursor miRNAs are trafficked along axons associated with vesicles and locally processed to regulate growth cone steering

Various species of non-coding RNAs (ncRNAs) are enriched in subcellular compartments but the mechanisms orchestrating their delocalization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked, anchored to CD63-positive vesicles, to distal axons along microtubules. Upon exposure to the chemotropic cue Sema3A, pre-miRNAs are processed specifically within axons into newly synthesized mature miRNAs, which, in turn, silence the basal translation of TUBB3 but not of APP. At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly synthesized miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodelling of subcellular compartments.\n\nHighlightsO_LIPrecursor miRNAs are actively transported along axons to the growth cone tethered to CD63-positive vesicles\nC_LIO_LISema3A but not Slit2 induces the local biogenesis of specific miRNAs within axons\nC_LIO_LIMature miRNAs are important for growth cone responsiveness ex vivo and the establishment of functional connections in vivo\nC_LIO_LINewly synthesized miRNAs inhibit the basal translation of TUBB3 but not APP upon Sema3A exposure\nC_LI

cell biology↗