Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

The hnRNP family: insights into their role in health and disease

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that contribute to multiple aspects of nucleic acid metabolism including alternative splicing, mRNA stabilization, and transcriptional and translational regulation. Many hnRNPs share general features, but differ in domain composition and functional properties. This review will discuss the current knowledge about the different hnRNP family members, focusing on their structural and functional divergence. Additionally, we will highlight their involvement in neurodegenerative diseases and cancer, and the potential to develop RNA-based therapies

Spatial mapping of splicing factor complexes involved in exon and intron definition

Nuclear pre-mRNA splicing occurs in eukaryotic cells in order to remove the intervening sequences (introns) that interrupt coding regions (exons) of genes. This pre-mRNA splicing occurs in the spliceosome (a quite large RNP complex), the assembly of which starts upon recognition of the 5′ and 3′ splice sites by the Ul small nuclear ribonucleoprotein (snRNP) and U2 snRNP auxiliary factor (U2AF), respectively. In addition to the small RNP particles Ul, U2, U4/U6, and U5 snRNPs, numerous non-snRNP splicing factors constitute the spliceosome, among which is the SR family of proteins, which have a modular domain structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RRMs determine RNA binding specificity, while the RS domain mediates protein-protein interactions. In this study, fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) techniques were used to determine protein-protein interaction between splicing factors in nanometer resolutions. The interaction of splicing factor 2/alternative splicing factor (SF2/ASF) with Ul 70K (the Ul snRNP-associated protein) in live HeLa cells was revealed via FRET acceptor photobleaching microscopy. After photobleaching, a strong FRET signal was detected in HeLa cells coexpressing ECFP-U1 70K and EYFP- SF2/ASF (Fig. 1), indicating that the two proteins can interact in vivo. Treatment with DRB, a transcription inhibitor, did not prevent Ul 70K and SF2/ ASF interaction in HeLa cells, demonstrating that their interaction does not occur exclusively upon pre-mRNA. SF2/ASF interaction with U2AF35 occurs as speckles in the nucleus of HeLa cells. The researchers have also shown that both SC35 and SRp20 interact with Ul 70K and U2AF35 in live HeLa cells. DRB treatment also did not prevent their interaction. A novel interaction of HCC1, a protein factor highly homologous to U2AF65, with U2AF35 and with U2AF65 was also revealed by Co-IP experiments and FRET microscopy.</p

Cooperation between C<i>aenorhabditis elegans</i>COMPASS and condensin in germline chromatin organization

AbstractDeposition of histone H3 lysine 4 (H3K4) methylation at promoters by SET1/COMPASS is associated with context-dependent effects on gene expression and local changes in chromatin organization. Whether SET1/COMPASS also contributes to higher-order chromosome structure has not been investigated. Here, we address this question by quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) onC. elegansgerm cells expressing histones H2B-eGFP and H2B-mCherry. We find that SET1/COMPASS subunits strongly influence meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of SET-2, theC. eleganshomologue of SET1, or CFP-1, the chromatin targeting subunit of COMPASS, strongly enhance chromosome organization defects and loss of fertility resulting from depletion of condensin-II. Defects in chromosome morphology resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes, were also aggravated in the absence of SET-2. Combined, ourin vivofindings suggest a model in which the SET1/COMPASS histone methyltransferase complex plays a role in shaping meiotic chromosome in cooperation with the non-histone proteins condensin-II and topoisomerase.</jats:p

Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice

International audienceHeterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an abundant nuclear protein that binds to pre-mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP-M and the human spliceosome proteins cell division cycle 5-like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat-shock stress response. A central region in hnRNP-M is required for interaction with CDC5L/PLRG1. hnRNP-M affects both 5 0 and 3 0 alternative splice site choices, and an hnRNP-M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno-E1A mini-gene substrate

(A) HeLa cells were cotransfected with EGFP-U2AF35 and either mCherry-C1 or mCherry-SF2/ASF

Confocal images are of transfected cells and FLIM images of the same cells, in which FRET efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-U2AF35 + mCherry-C1; Middle, EGFP-U2AF35 + mCherry-SF2/ASF; Bottom, EGFP-U2AF35 + mCherry-SF2/ASF in the presence of DRB. Bars, 10 μm. (B) FRET efficiencies determined by FLIM for interaction of SF2/ASF with U2AF35 in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for seven to nine cells. To measure the FRET efficiency in the speckles and nucleoplasm, a region characteristic of each was selected for each cell. P-values were obtained as described in the legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) HeLa cells were transfected with EGFP–U1 70K and cotransfected with either mCherry-C1 or mCherry-SF2/ASF

Shown are confocal images of transfected cells and FLIM images of the same cells, in which mean fluorescence lifetime is shown in pseudocolor. The color scale with the respective lifetimes (in picoseconds [ps]) is indicated. The percentage of FRET efficiencies and FRET amplitude are shown in continuous pseudocolor. The color scale with the respective FRET efficiencies (percentage) is indicated. The FRET amplitude % represents the fraction of interacting donor molecules, also defined as the FRET population % (or concentration of FRET species). (B) FRET between U1 70K and SF2/ASF, in the presence of DRB, measured by FLIM. Experiments were performed exactly as described for A, except cells were treated with 25 μg/ml of DRB for 2 h before images were taken. Bars, 10 μm. (C) FRET efficiencies calculated from FLIM measurements for the interaction of SF2/ASF with U1 70K in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for 9–20 cells. To measure the FRET efficiency in the speckles and nucleoplasm, a region characteristic of each was selected for each cell. P-values were obtained as described in the legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p