68 research outputs found
Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines
We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2
Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library
Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress
RNA Modulators of Complex Phenotypes in Mammalian Cells
RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-α)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators
Enhanced snoMEN Vectors Facilitate Establishment of GFPâHIF-1α Protein Replacement Human Cell Lines
The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFPâHIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy
Ectopic synaptic ribbons in dendrites of mouse retinal ON- and OFF-bipolar cells
The ectopic distribution of synaptic ribbons in dendrites of mouse retinal bipolar cells was examined by using genetic ablation of metabotropic glutamate receptor subtype 6 (mGluR6), electron microscopy, and immunocytochemistry. Ectopic ribbons were observed in dendrites of rod and ON-cone bipolar cells in the mGluR6-deficient mouse but not in those of wild-type mice. The number of rod spherules facing the ectopic ribbons in mGluR6-deficient rod bipolar dendrites increased gradually during early growth and reached a plateau level of about 20% at 12Â weeks. These ectopic ribbons were immunopositive for RIBEYE, a ribbon-specific protein, but the associated vesicles were immunonegative for synaptophysin, a synaptic-vesicle-specific protein. The presence of ectopic ribbons was correlated with an increase in the roundness of the invaginating dendrites of the rod bipolar cells. We further confirmed ectopic ribbons in dendrites of OFF-cone bipolar cells in wild-type retinas. Of the four types of OFF-cone bipolar cells (T1âT4), only the T2-type, which had a greater number of synaptic ribbons at the axon terminal and a thicker axon cylinder than the other types, had ectopic ribbons. Light-adapted experiments revealed that, in wild-type mice under enhanced-light adaptation (considered similar to the mGluR6-deficient state), the roundness in the invaginating dendrites and axon terminals of rod bipolar cells increased, but no ectopic ribbons were detected. Based on these findings and known mechanisms for neurotransmitter release and protein trafficking, the possible mechanisms underlying the ectopic ribbons are discussed on the basis of intracellular transport for the replenishment of synaptic proteins
FMN2 is a novel regulator of the cyclin-dependent kinase inhibitor p21
We have identified the human FMN2 gene as a novel target regulated by induction of p14ARF and by multiple other stress responses, including DNA damage and hypoxia, which have in common activation of cell cycle arrest. We showed that increased expression of the FMN2 gene following p14ARF induction is caused, at the transcriptional level, by relief of repression by RelA and E2F1, which, under non-induced conditions, bind the FMN2 promoter. Increased FMN2 protein levels promote cell cycle arrest by inhibiting the degradation of p21, and our data show that control of p21 stability is a key part of the mechanism that regulates p21 induction. Consistent with this model, we have shown that transient expression of exogenous FMN2 protein alone is sufficient to increase p21 protein levels in cells, without altering p21 mRNA levels. Here, we provide additional evidence for the role of the N terminus of FMN2 as being the important domain required for p21 stability. In addition, we also investigate the role of RelAâs threonine 505 residue in the control of FMN2. Our results identify FMN2 as a crucial protein involved in the control of p21
Features of snoMEN technology.
<p>Schematic diagram showing differences between the siRNA/shRNA and snoMEN systems. Arrows show promoters for RNA polymerase III (shRNA) and RNA polymerase II (snoMEN), respectively. Red squares show the coding region, e.g. either mCherry cDNA, or endogenous genes. Striped squares show non-coding exon region. The bars show non-coding regions, e.g. introns.</p
Potential factors involved in snoMEN machinery.
<p>(<b>A</b>) U2OS<sup>GFPâSMN1-PR</sup> and U2OS<sup>GFPâSMN1</sup> cells were transfected with either Scrambled siRNA, Ago1 siRNA, Ago2 siRNA, Upf1 siRNA, or fibrillarin siRNA. An equivalent amount of each extract was loaded in each lane and the proteins were separated by SDS PAGE, electroblotted onto membrane and probed with anti-SMN1, anti-Ago1, anti-Ago2, anti-Upf1, anti-fibrillarin and with anti-tubulin as a loading control. (<b>B</b>) The graphs show average SMN signal intensity and standard deviation for three independent experiments using the same procedure as in A. SMN signal ratio was normalised to the tubulin signal.</p
Characterisation of FPâprotein complexes in replacement stable cell lines by Quantitative SILAC Proteomic analysis.
<p>(<b>A</b>) Design of the triple-encoding SILAC pull down experiments (see text). Comparison of mCherryâUBF1 complex, either in the presence, or absence, of low concentration Actinomycin-D (left panel) and comparison of GFPâSMN1 complex either with replacement (U2OS<sup>GFPâSMN1-PR</sup>), or without replacement (U2OS<sup>GFPâSMN1</sup>) (right panel). The SILAC experiments were independently repeated at least four times. (<b>B</b>) SILAC result of mCherryâUBF1 complex pull down assay visualised on a 2D logarithmic graph for all proteins identified. On the x axis, log<sub>2</sub> (M/L ratio) correlates with the enrichment in mCherryâUBF1 IP versus control IP. On the y axis, log<sub>2</sub> (H/L ratio) correlates with the enrichment in mCherryâUBF1 IP with Actinomycin-D treatment versus no-treatment IP. The bait, UBF1, is shown in red, and a blue line separates the proteins whose interaction with UBF1 is increased (above the line)/or decreased (below) after Actinomycin-D treatment. Known interaction partners, which were identified and quantified, are also highlighted. SILAC ratio values of labelled proteins are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062305#pone.0062305.s007" target="_blank">Table S2</a>. (<b>C</b>) Known UBF1 interaction partners, which were identified and quantified. Graph shows fold change of each protein ratio with Actinomycin-D treatment (red) and without treatment (orange) measured from five independent experiments. (<b>D</b>) Comparisons of protein ratios of known interaction partners of SMN1 between over expression stable cell line (U2OS<sup>GFPâSMN1</sup>; light green) and protein replacement stable cell line (U2OS<sup>GFPâSMN1-PR</sup>; green) measured from five independent experiments.</p
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