Showing papers by "Phillip A. Sharp published in 2002"

Gene silencing in mammals by small interfering RNAs

Abstract: Among the 3 billion base pairs of the human genome, there are approximately 30,000-40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool - short interfering RNAs (siRNAs) - has now been developed for systematically deciphering the functions and interactions of these thousands of genes. siRNAs are an intermediate of RNA interference, the process by which double-stranded RNA silences homologous genes. Although the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, the emerging literature indicates that most vertebrate genes can be studied with this technology.

Predictive Identification of Exonic Splicing Enhancers in Human Genes

Abstract: Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing. A computational method, RESCUE-ESE, was developed that predicts which sequences have ESE activity by statistical analysis of exon-intron and splice site composition. When large data sets of human gene sequences were used, this method identified 10 predicted ESE motifs. Representatives of all 10 motifs were found to display enhancer activity in vivo, whereas point mutants of these sequences exhibited sharply reduced activity. The motifs identified enable prediction of the splicing phenotypes of exonic mutations in human genes.

siRNA-directed inhibition of HIV-1 infection.

Abstract: RNA interference silences gene expression through short interfering 21 23-mer double-strand RNA segments that guide mRNA degradation in a sequence-specific fashion. Here we report that siRNAs inhibit virus production by targeting the mRNAs for either the HIV-1 cellular receptor CD4, the viral structural Gag protein or green fluorescence protein substituted for the Nef regulatory protein. siRNAs effectively inhibit pre- and/or post-integration infection events in the HIV-1 life cycle. Thus, siRNAs may have potential for therapeutic intervention in HIV-1 and other viral infections.

Gene silencing using micro-RNA designed hairpins.

Abstract: During RNA interference (RNAi), long dsRNA is processed to ;21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are ;21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.

Small Interfering RNA-Mediated Gene Silencing in T Lymphocytes

Abstract: Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8α specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.

Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro

Abstract: Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) II is ubiquitinated in cells after UV-irradiation or cisplatin treatment, which induces DNA damage preferentially repaired by TCR. Several human mutations, such as Cockayne syndrome complementation groups A and B, are defective in TCR and incapable of Pol II ubiquitination upon DNA damage. Here we demonstrate a correlation between ubiquitination of RNA Pol II and arrest of transcription in vitro. Ubiquitination of Pol II is significantly induced by α-amanitin, an amatoxin that blocks Pol II elongation and causes its degradation in cells. Pol II undergoes similar ubiquitination on DNA containing cisplatin adducts that arrest transcription. Stimulation of ubiquitination requires the addition of template DNA and does not occur in the presence of an antibody to the general transcription factor TFIIB, indicating the transcription dependence of the reaction. We propose that components of the reaction recognize elongating Pol II–DNA complexes arrested by α-amanitin or cisplatin lesions, triggering ubiquitination.

Decreased expression of the vitamin C transporter SVCT1 by ascorbic acid in a human intestinal epithelial cell line.

Abstract: Vitamin C (ascorbic acid) is an essential nutrient that is involved in a number of cellular processes. However, unlike most mammals, man is unable to synthesize vitamin C and it must therefore be acquired from the diet. Absorption of vitamin C is achieved by two transporters, SVCTI and SVCT2, recently cloned from rat and human kidney. SVCT1 is thought to be the predominant transporter in the intestine. Vitamin C supplements are increasingly common, thus contributing to an increased dietary load, and therefore the aim of the present study was to investigate the effect of high doses of ascorbic acid on SVCT1 expression. Using the Caco-2 TC7 cell model of small intestinal enterocytes, we measured the effects of ascorbic acid (4.5 mg/ml culture medium) on L-[14C]ascorbic acid uptake and SVCT1 expression (determined by reverse transcription-polymerase chain reaction). Ascorbic acid uptake was decreased significantly in Caco-2 TC7 cells exposed to ascorbate for 24 h (-50%, P<0.0005). Expression of SVCT1 was also significantly reduced by exposure to elevated levels of ascorbate for 24h (-77%, P<0.005). Taken together these results suggest that high-dose supplements might not be the most efficient way of increasing the body pool of vitamin C.

PDCD2 is a negative regulator of HCF-1 (C1).

Abstract: Temperature sensitive mutations in host cell factor 1 (HCF-1) arrest cells in the middle of the G1 phase of the cycle. We have shown that the highly conserved C-terminal WYF domain of HCF-1 protein interacts with the MYND domain of the PDCD2 protein. This inter-action is conserved between human HCF-1 and HCF-2 and the C. elegans HCF. Overexpression of PDCD2, which interacts with the N-CoR/mSin3A corepressor complexes, suppresses cotransfected HCF-1 complement-ation of a temperature lesion in the endogenous HCF-1 protein. Overexpression of domains of either PDCD2 or HCF-1, which should interfere with interactions between these two proteins, enhances the complementation.

The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

Abstract: 1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.

Mouse lymphoid cell line selected to have high immunoglobulin promoter activity

Abstract: Immunoglobulin variable region promoters are predominantly B-cell specific, but the molecular basis for this specificity has not been elucidated. To further understand how B-cell-specific immunoglobulin promoter expression is mediated, the murine lymphoid cell line 2017 was engineered to express the green fluorescent protein under the control of an immunoglobulin heavy chain promoter and selected for high activity using multiple rounds of fluorescence-activated cell sorting. Rare clones with intense and stable immunoglobulin promoter activity were isolated. Transient transfection experiments demonstrated that two different immunoglobulin promoters and two other B-cell-specific promoters have higher activities in the selected cell lines relative to the parental line and to the non-cell-type-specific histone H2B promoter. The increased immunoglobulin activity required nucleotide residues downstream of the transcription initiation site which were also important for maximal activity in B cells and which were conserved in other B-cell-specific promoters. Unlike the unselected cells, the 2017 variants also showed activation of their endogenous immunoglobulin heavy chain variable regions.