Identification of the ‘NORE’ (N-Oct-3 responsive element), a novel structural motif and composite element

N-Oct-3 is a neuronal transcription factor widely expressed in the developing mammalian central nervous system, and necessary to maintain neural cell differentiation. The key role of N-Oct-3 in the transcriptional regulation of a multiplicity of genes is primarily due to the structural plasticity of its so-called ‘POU’ (acronym of Pit, Oct, Unc) DNA-binding domain. We have recently reported about the unusual dual neuro-specific transcriptional regulation displayed by N-Oct-3 [Blaud,M., Vossen,C., Joseph,G., Alazard,R., Erard,M. and Nieto,L. (2004) J. Mol. Biol., 339, 1049–1058]. To elucidate the underlying molecular mechanisms, we have now made use of molecular modeling, DNA footprinting and electrophoretic mobility shift assay techniques. This combined approach has allowed us to uncover a novel mode of homodimerization adopted by the N-Oct-3 POU domain bound to the neuronal aromatic amino acids de-carboxylase and corticotropin-releasing hormone gene promoters and to demonstrate that this pattern is induced by a structural motif that we have termed ‘NORE’ (N-Oct-3 responsive element), comprising the 14 bp sequence element TNNRTAAATAATRN. In addition, we have been able to explain how the same structural motif can also induce the formation of a heterodimer in association with hepatocyte nuclear factor 3β(/Forkhead box a2). Finally, we discuss the possible role of the NORE motif in relation to neuroendocrine lung tumor formation, and in particular the development of small cell lung cancer

Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C- domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins

RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA: Psi-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature

International audienceThe box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Psi) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyper-thermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to 111 conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity. (C) 2015 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved

Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity

International audienceMultiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21

Chaperoning 5S RNA assembly

International audienc

Different conformational changes of sRNA Pab21 and Pab91 are associated with the sequential binding of the sRNP proteins.

<p>(<b>A</b>) CD spectra of sRNAs Pab21 (red) and Pab91 (blue) scanned in the range 240 to 320 nm. (<b>B</b>) CD spectra of sRNA Pab21 obtained when protein aCBF5 and the sRNA are present in two separate cell compartments (dark blue) or after mixing the two compartments (green). Spectra obtained following sequential addition of proteins to the aCBF5–Pab21 complex: aNOP10 (magenta) added first and then L7Ae (light blue). (<b>C</b>) Same experiment as in panel B but with Pab91. (<b>D</b>) CD spectra of sRNA Pab21 obtained with the sRNA and L7Ae in separate compartments (dark purple) and after mixing (purple). (<b>E</b>) Quantification of the decrease in the peak amplitude of the specific RNA positive signal at the λ_max wavelength, generated exclusively by addition of L7Ae to different aCBF5–aNOP10–sRNA complexes. The resulting δ(Δε) values are displayed as a histogram for each complex. sRNP assembly was achieved by incubation of aCBF5–Pab21 with the mutant of L7Ae (H70A/L74A/E77A designated HLE) or the mutant of aNOP10 (Y41A/Y44A designated Y).</p

Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probe

International audienceNeutrophil granules (NGs) are key components of the innate immune response and mark the development of the hematopoietic system in mammals. However, no specific fluorescent vital stain existed up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe (HAB) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell-and organelle-specific small-molecule tracer of NGs in live zebrafish larvae, with no labeling of any other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically-active cells with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB-stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosome in live zebrafish phagocytosing neutrophils. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically-relevant competing chalcones, indicating that this specific labeling was the result of HAB precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically-incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems

Swapping of helix P1 between sRNAs Pab21 and Pab91.

<p>(A) Scheme of the chimera obtained upon swapping the P1 basal helices. To obtain Pab91P₁21, sequences spanning from positions 9 to 17 and sequences from positions 54 to 62 in Pab91 were respectively substituted by sequences of Pab21 spanning from positions 7 to 15 and from positions 54 to 62. To obtain Pab21P₁91, sequences spanning from positions 7 to 15 and sequences from positions 54 to 62 in Pab21 were respectively substituted by sequences of Pab91 spanning from positions 9 to 17 and from positions 54 to 62. (<b>B</b>) EMSA of the sub-complex RNP2 formed upon incubation of the radiolabeled sRNAs (50 fmol) with various concentrations of the protein aCBF5 (50 to 400 nM) indicated on top of each lane. The amounts of radioactivity in the bands were estimated with the ImageQuant software. The percentage of RNA in each RNP was calculated from the radioactivity in each band relative to the total radioactivity in the lane. The estimated value of the apparent dissociation constant (K_D) is indicated for each aCBF5-sRNA complex. (<b>C</b>) CD spectra of the parental Pab21 and Pab91 and the chimeric sRNAs. Experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070313#pone-0070313-g004" target="_blank">Figure 4A</a>. (<b>D</b>) Time course analyses of the Ψ formation by the LCN RNPs in the substrate RNAs of Pab21 (Pab21 and Pab21P₁91) and of Pab91 (Pab91 and Pab91P₁21). Experimental conditions were the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070313#pone-0070313-g001" target="_blank">figure 1C</a>. After T2 RNase digestion, the amount of Ψ formation was estimated by 1D-TLC analysis. The wild type sRNAs Pab91 and Pab21 are represented by solid lines and full symbols, while the chimeric sRNAs Pab91P₁21 and Pab21P₁91 are represented by dashed lines and empty symbols.</p

Effect of mutations of residues lying at the aNOP10–L7Ae interface on H/ACA sRNP assembly and activity.

<p>(<b>A</b>) Secondary structure model of <i>P. abyssi</i> Pab21 and Pab91 sRNAs and of the interaction between the sRNAs and their respective substrate RNA (S). The ACA sequence at the 3′ end is boxed. The strands of the various motifs are indicated: KTa and KTb of the apical K-loop motif, P2a and P2b of helix P2, and P1a and P1b of helix P1. Helices SH1 and SH2 are formed upon interaction of the substrate RNA with the two strands s1 and s2 of the pseudouridylation pocket. (<b>B</b> and <b>C</b>) Electrophoretic mobility shift assay (EMSA) of the various sub-complexes formed upon incubation of the radiolabeled sRNAs (50 fmol) Pab21 (<b>B</b>) or Pab91 (<b>C</b>) with various combinations of the wild-type and mutant proteins (200 nM each). (<b>D</b>) Time course analyses of the RNA-guided Ψ formation in the substrate RNAs, which were radiolabeled during <i>in vitro</i> transcription by incorporation of [α-³²P]UTP for Pab21 substrate and of [α-³²P]CTP for Pab91 substrate. Each substrate RNA was incubated at 65°C with the unlabeled guide sRNA, and the protein sets aCBF5–aNOP10 (CN), L7Ae–aCBF5–aNOP10 (LCN), or L7Ae–aCBF5–aNOP10–aGAR1 (LCNG). A mutant of L7Ae (triple mutant H70A/L74A/E77A, designated HLE), or a mutant of aNOP10 Y41A/Y44A (designated Y) were used for the reaction. After T2 RNase digestion, the amount of Ψ formation was estimated by 1D-TLC analysis. (<b>E</b>) Close-up view of the contact region between L7Ae (displayed in gold) and aNOP10 (in green). Residues conserved between <i>P. abyssi</i> and <i>P. furiosus</i> are indicated on the <i>P. furiosus</i> sRNP structure (PDB 2HVY). Residues in red were substituted by alanine. (<b>F</b> and <b>G</b>) Analysis by EMSA of the complexes formed between the wild-type and mutant Pab21 sRNP (<b>F</b>) and Pab91 sRNP (<b>G</b>) and their respective radiolabeled substrate RNA. The yield of complex CII’ is indicated below each lane; the percentage of sRNA present in each CII′ complex was estimated by radioactivity measurement. The yields are expressed relative to that obtained with wild type sRNP (set to 100).</p