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Papers by Pascale Legault

F1000 - Post-publication peer review of the biomedical literature, 2015

RNA-mediated gene silencing in human cells requires the accurate generation of ∼22-nucleotide mic... more RNA-mediated gene silencing in human cells requires the accurate generation of ∼22-nucleotide microRNAs (miRNAs) from double-stranded RNA substrates by the endonuclease Dicer. Although the phylogenetically conserved RNA-binding proteins TRBP and PACT are known to contribute to this process, their mode of Dicer binding and their genome-wide effects on miRNA processing have not been determined. We solved the crystal structure of a human Dicer-TRBP interaction complex comprising two domains of previously unknown structure. Interface residues conserved between TRBP and PACT show that the proteins bind to Dicer in a similar manner and by mutual exclusion. Based on the structure, a catalytically active Dicer that cannot bind TRBP or PACT was designed and introduced into Dicer-deficient mammalian cells, revealing selective defects in guide strand selection. These results demonstrate the role of Dicer-associated RNA binding proteins in maintenance of gene silencing fidelity.

p65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the ... more p65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the activating component of the p65–p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAFII31. Like other acidic TADs, the p65 TAD contains two subdomains (p65TA1 and p65TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-κB target genes, there are no high-resolution structures of p65TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65TA1 transitions into a helical conformation that includes its characteristic ΦXXΦΦ motif (Φ = hydrophobic amino acid). Structural and functional studies de...

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

ABSTRACTIn Parkinson’s disease, the most vulnerable neurons are found in the ventral tier of the ... more ABSTRACTIn Parkinson’s disease, the most vulnerable neurons are found in the ventral tier of the substantia nigra (SN), while the adjacent dopamine (DA) neurons of the ventral tegmental area (VTA) are mostly spared. Although a significant subset of adult VTA DA neurons expresses Vglut2, a vesicular glutamate transporter, and release glutamate as a second neurotransmitter in the striatum, only very few adult SN DA neurons have this capacity. Previous work has demonstrated that lesions created by neurotoxins such as MPTP and 6-hydroxydopamine (6-OHDA) can upregulate the expression of Vglut2 in surviving DA neurons. Currently, the molecular mechanisms explaining the plasticity of Vglut2 expression in DA neurons are unknown, as are the physiological consequences for DA neuron function and survival. Here we aimed to characterize the developmental expression pattern of Vglut2 in DA neurons and the role of this transporter in post-lesional plasticity in these neurons. Using an intersection...

Nucleic Acids Research

Investigating the dynamics of structural elements in functional RNAs is important to better under... more Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.

BMC Biotechnology

Background: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as t... more Background: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure. Results: Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure (≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K d) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k cat (7.2 ± 0.5 min − 1) and K M (1.2 ± 0.3 μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. Conclusions: The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k cat and K M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

F1000 - Post-publication peer review of the biomedical literature

Nucleic Acids Research

p65 is a member of the NF-B family of transcriptional regulatory proteins that functions as the a... more p65 is a member of the NF-B family of transcriptional regulatory proteins that functions as the activating component of the p65-p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAF II 31. Like other acidic TADs, the p65 TAD contains two subdomains (p65 TA1 and p65 TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-B target genes, there are no high-resolution structures of p65 TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65 TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65 TA1 transitions into a helical conformation that includes its characteristic XX motif (= hydrophobic amino acid). Structural and functional studies demonstrate that the two binding interfaces are primarily stabilized by three hydrophobic amino acids within the XX motif and these residues are also crucial to its ability to activate transcription. Taken together, the results provide an atomic level description of how p65 TA1 is able to bind different transcriptional regulatory factors needed to activate NF-B target genes.

Wiley interdisciplinary reviews. RNA, Jan 6, 2017

Despite the large number of noncoding RNAs and their importance in several biological processes, ... more Despite the large number of noncoding RNAs and their importance in several biological processes, our understanding of RNA structure and dynamics at atomic resolution is still very limited. Like many other RNAs, the Neurospora Varkud satellite (VS) ribozyme performs its functions through dynamic exchange of multiple conformational states. More specifically, the VS ribozyme recognizes and cleaves its stem-loop substrate via a mechanism that involves several structural transitions within its stem-loop substrate. The recent publications of high-resolution structures of the VS ribozyme, obtained by NMR spectroscopy and X-ray crystallography, offer an opportunity to integrate the data and closely examine the structural and dynamic properties of this model RNA system. Notably, these investigations provide a valuable example of the divide-and-conquer strategy for structural and dynamic characterization of a large RNA, based on NMR structures of several individual subdomains. The success of ...

RNA, 2016

As part of their normal life cycle, most RNA molecules associate with several proteins that direc... more As part of their normal life cycle, most RNA molecules associate with several proteins that direct their fate and regulate their function. Here, we describe a novel method for identifying proteins that associate with a target RNA. The procedure is based on the ARiBo method for affinity purification of RNA, which was originally developed to quickly purify RNA with high yields and purity under native conditions. The ARiBo method was further optimized using in vitro transcribed RNA to capture RNA-associating proteins from cellular extracts with high yields and low background protein contamination. For these RNA pull-downs, stem–loops present in the immature forms of let-7 miRNAs (miRNA stem–loops) were used as the target RNAs. Label-free quantitative mass spectrometry analysis allowed for the reliable identification of proteins that are specific to the stem–loops present in the immature forms of two miRNAs, let-7a-1 and let-7g. Several proteins known to bind immature forms of these let...

Nucleic Acids Research, Feb 28, 2012

F1000 - Post-publication peer review of the biomedical literature, 2014

F1000 - Post-publication peer review of the biomedical literature, 2012

F1000 - Post-publication peer review of the biomedical literature, 2009

F1000 - Post-publication peer review of the biomedical literature, 2015

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

Nucleic Acids Research

BMC Biotechnology

F1000 - Post-publication peer review of the biomedical literature

Nucleic Acids Research

Wiley interdisciplinary reviews. RNA, Jan 6, 2017

RNA, 2016

Nucleic Acids Research, Feb 28, 2012

F1000 - Post-publication peer review of the biomedical literature, 2014

F1000 - Post-publication peer review of the biomedical literature, 2012

F1000 - Post-publication peer review of the biomedical literature, 2009