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At least 415 records · Page 23Linked to original sources
PubMed2003

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA.

Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking G beta(2) but is unaffected in cells lacking G beta(1), G alpha i(2), or G alpha i(3). Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by G beta(2) knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking G beta(1). Cells lacking G beta(2) retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking G beta(2) can be reversed by overexpression of either human G beta(2) or mouse G beta(1). These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, G beta(2)-derived G beta gamma is the most effective mediator of chemotaxis to C5a.

Animals↗
PubMed1995

Accurate transcription of the Trypanosoma brucei U2 small nuclear RNA gene in a homologous extract.

In vitro transcription systems are a classic means to dissect mechanisms of gene expression at the molecular level. To begin an analysis of the biochemistry of gene expression in trypanosomes, we established an in vitro transcription system from cultured insect forms of Trypanosoma brucei. As a model we used the U2 snRNA gene which in vivo is transcribed by an RNA polymerase with characteristics of animal RNA polymerase III. To obtain maximum sensitivity in our assay, we adapted the so-called G-less cassette approach to the U2 snRNA gene promoter. Since an intragenic control region is required for accurate expression in vivo, we generated a series of mutations to substitute all guanosine residues in the intragenic control region. These mutants were shown to retain full transcriptional activity in vivo after transient expression in insect-form trypanosomes. In a cell-free extract, synthesis of the U2 G-less cassette RNA is correctly initiated, is mediated by RNA polymerase III as determined by RNA polymerase inhibitor studies, and is dependent on the integrity of the upstream B box element.

Animals↗
PubMed2003

Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA. Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide.

Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110alpha isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA). This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110alpha. Notably, p110alpha was silenced without affecting levels of either the other class I(A) PI3K catalytic subunits p110beta and p110delta, or the p85alpha regulatory subunit. The role of p110alpha in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1alpha,25-dihydroxycholecalciferol (D(3)) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D(3) was sensitive to silencing of p110alpha, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D(3) in a PI3K-dependent manner and gene silencing using shRNA showed that p110alpha was also required for this effect. Taken together, these findings demonstrate that LPS and D(3) use distinct isoforms of class I(A) PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.

Calcitriol↗
PubMed2003

Specific killing of Ph+ chronic myeloid leukemia cells by a lentiviral vector-delivered anti-bcr/abl small hairpin RNA.

Chronic myeloid leukemia (CML) is characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 t(9;22)(q34;q11) that causes fusion of the bcr and abl genes. Transcription and splicing of the fusion gene generate two major splice variants of the bcr/abl transcript that encode an oncoprotein with tyrosine kinase activity. We have taken advantage of lentiviral vectormediated delivery of anti-bcr/abl short hairpin RNAs (shRNA) to downregulate the bcr/abl transcript in Philadelphia chromosome-positive (Ph+) K562 leukemia cells. This downregulation caused complete inhibition of proliferation of these cells and was accompanied by >90% inhibition of the bcr/abl transcript and p210 protein. These results demonstrate the feasibility of using a lentiviral vector to stably transduce therapeutic shRNAs into leukemia cells for the potential ex vivo purging of Ph+ cells in an autologous hematopoietic cell transplant setting. Furthermore, the robust expression of the shRNAs from our lentiviral vector suggests that this system could be generally useful for the expression of other shRNAs.

Base Sequence↗
PubMed2004

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay.

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.

Calcium-Binding Proteins↗
PubMed2004

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not.

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

Actins↗
PubMed1984

Pseudogenes for human U2 small nuclear RNA do not have a fixed site of 3' truncation.

We present the sequences of five additional human U2 pseudogenes which are very similar to the U2.13 pseudogene reported previously [Van Arsdell et al. (1981) Cell 26, 11-17]. All six U2 pseudogenes preserve the 5' end of the mature U2 snRNA sequence, and all six are flanked by nearly perfect direct repeats that differ in sequence and range in length from 16 to 21 base pairs. The 3' ends of the six U2 pseudogenes are truncated at five different sites between position 33 and 82, and in two cases the 3' end of the pseudogene overlaps the downstream direct repeat by 5 or 6 base pairs. The structure of these six U2 pseudogenes contrasts with that of four human U3 pseudogenes [Bernstein et al. (1983) Cell 32, 461-472] all of which are identically truncated at position 69 or 70, and appear to be derived from a self-primed 74 base reverse transcript of U3 snRNA. Comparison of the U2 and U3 pseudogenes suggests a model for their generation in which the 3' end of the pseudogene is always truncated relative to the initial cDNA template.

Bacteriophage lambda↗
PubMed1987

Identification of proteins interacting with the enhancer of human U2 small nuclear RNA genes.

Protein/DNA interactions in the human U2 RNA gene enhancer have been characterized by DNase I footprint and DMS methylation protection analyses. Nuclear factors present in both HeLa and B cell extracts have been shown to protect an approximately 70 bp region from DNase I digestion. DMS and DNase I footprint competition studies demonstrated that the entire footprint can be accounted for by interactions with two previously identified transcription factors. One of these recognizes the so called octanucleotide-motif ATGCAAAT (transcription factor IgNF-A) which has been shown to be essential for transcription. The other is the transcription factor Sp1 which binds to three target sequences located adjacent to the octameric motif. The Sp1 interactions appear to be required for full transcriptional activity. No differences in the DNase I footprint patterns or in the DMS methylation protections were observed when nuclear extracts from HeLa cells, two different B cell lines, or from the adenovirus-transformed 293 cell line were compared.

Base Sequence↗
PubMed1992

The transcriptional start site for a human U6 small nuclear RNA gene is dictated by a compound promoter element consisting of the PSE and the TATA box.

Transcription of vertebrate U6 snRNA genes by RNA polymerase III requires two sequence elements in the proximal promoter region: the PSE (proximal sequence element, found in snRNA promoters transcribed by RNA polymerase II) and the TATA element (found in many mRNA promoters). The locations of the PSE and the TATA box are important determinants for transcriptional start site selection in their respective RNA polymerase II promoters. In vertebrate U6 genes the PSE and the TATA elements are located in approximately the same positions as in the polymerase II transcribed genes, but their respective roles in initiation site selection are unknown. We have analyzed the effects of spacing changes between the PSE and the TATA element, and between the two elements and the normal U6 start site on human U6 gene transcription. The spacing requirement between the two elements is highly stringent, implying a possible interaction between the factors that bind them. Our results discount the possibility that the location of either the PSE or the TATA element, by itself, dictates efficient selection of a transcriptional start site. Instead, we suggest that the two elements form a compound promoter element whose location dictates the start site of transcription from the human U6 gene promoter.

Base Sequence↗
PubMed1992

Genes for Xenopus laevis U3 small nuclear RNA.

Genomic Southern blots showed there are only 14 to 20 copies of U3 snRNA genes per somatic genome in Xenopus laevis, unlike the highly repetitive, tandem arrangement of other snRNA genes in this organism. Sequencing of two U3 snRNA genes from lambda clones of a genomic library revealed striking similarity upstream, but much more divergence downstream. Consensus motifs common to other U snRNA genes were also found: a distal sequence element (DSE, octamer motif at -222 to -215), a proximal sequence element (PSE, at -62 to -52) and a 3' Box (15 or 16 bp downstream of the U3 genes). The DSE of mammals also has an inverted CCAAT motif specific for U3 snRNA genes, and we find this is conserved in the amphibian U3 snRNA genes. The Xenopus inverted CCAAT motif is exactly one helical turn further upstream of the octamer motif than its mammalian counterpart, suggesting interaction of putative transcription factors bound to these motifs. Mutation of the inverted CCAAT motif and part of an adjacent Sp1 site greatly depresses transcription of the mutant U3 snRNA gene in Xenopus oocytes, implying a role in transcriptional efficiency. Electrophoretic mobility shift assays implicate transcription factor binding to this region.

Animals↗
PubMed1994

U21, a novel small nucleolar RNA with a 13 nt. complementarity to 28S rRNA, is encoded in an intron of ribosomal protein L5 gene in chicken and mammals.

Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.

Animals↗