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PubMed2002

Identification and characterization of a novel U14 small nucleolar RNA gene cluster in Oryza sativa.

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. Through computer search in international rice genome database, a novel U14 snoRNA gene cluster, consisting of two U14 snoRNA gene candidates, was found on rice chromosome II. They both have box C/D sequences and a 14 nucleotides (nt)-long complementarity to rice 18S ribosomal RNA (rRNA). Functional analysis of this gene cluster indicated that both were transcribed in vivo and might guide the methylations of C418 in rice 18S rRNA. By using primer extension, 5' and 3' rapid amplification of cDNA ends, the 5' and 3' ends of two snoRNAs were determined. The 52 nt long intergenic spacer of the gene cluster is rich in uridine. The absence of a conserved promoter element in this spacer, the proximity of the genes and the detection of transcripts containing linked U14 snoRNAs by reverse transcript polymerase chain reaction suggest that the rice U14 snoRNAs encoded in the cluster are transcribed as a polycistron under an upstream promoter, and individual U14 snoRNAs are released after processing of the precursor RNAs.

Base Sequence↗
PubMed1992

Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNA.

A class of RNA-containing particles, U small nuclear/nucleolar ribonucleoprotein particles (U snRNP), are well known to be targets for sera from patients with various autoimmune diseases. In the most cases the protein components carry the antigenic determinants. We have identified serum autoantibodies from three patients with systemic sclerosis that were directed against U1-U5 snRNA by immunoprecipitation of deproteinized 32PO4 labeled HeLa cell total RNA. By competitive radioimmunoprecipitation assays, an experimentally induced anti-2,2,7-trimethylguanosine (TMG) cap structure mAb inhibited the reaction of these antisera. In addition, IgG isolated from the antisera inhibited the anti-TMG mAb reaction to the U snRNA. Furthermore, a structural analog, 7-methylguanosine-triphosphate, competitively inhibited the reaction of the antisera to the U snRNA. Thus we concluded that the TMG cap structure of the U snRNA could be a target for serum autoantibodies.

Autoantibodies↗
PubMed1984

Precursors of U4 small nuclear RNA.

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.

Antibodies, Monoclonal↗
PubMed2003

Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action.

In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNA-directed mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein. Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.

Animals↗
PubMed2001

A novel brain-specific box C/D small nucleolar RNA processed from tandemly repeated introns of a noncoding RNA gene in rats.

Antisense box C/D small nucleolar RNAs (snoRNAs) guide the 2'-O-ribose methylations of eukaryotic rRNAs and small nuclear RNAs (snRNAs) through formation of a specific base pairing at each RNA methylation site. By analysis of a box C/D snoRNA cDNA library constructed from rat brain RNAs, we have identified a novel box C/D snoRNA, RBII-36, which is devoid of complementarity to rRNA or an snRNA and exhibits a brain-specific expression pattern. It is uniformly expressed in all major areas of adult rat brain (except for choroid plexus) and throughout rat brain ontogeny but exclusively detected in neurons in which it exhibits a nucleolar localization. In vertebrates, known methylation guide snoRNAs are intron-encoded and processed from transcripts of housekeeping genes. In contrast, RBII-36 snoRNA is intron-encoded in a gene preferentially expressed in the rat central nervous system and not in proliferating cells. Remarkably, this host gene, which encodes a previously reported noncoding RNA, Bsr, spans tandemly repeated 0.9-kilobase units including the snoRNA-containing intron. The novel brain-specific snoRNA appears to result not only from processing of the debranched lariat but also from endonucleolytic cleavages of unspliced Bsr RNA (i.e. an alternative splicing-independent pathway unreported so far for mammalian intronic snoRNAs). Sequences homologous to RBII-36 snoRNA were exclusively detected in the Rattus genus of rodents, suggesting a very recent origin of this brain-specific snoRNA.

Animals↗
PubMed2005

Small interfering RNA for experimental cancer therapy.

RNA interference describes the recently discovered process of sequence-specific, post-transcriptional gene silencing that is initiated by double-stranded RNA molecules known as small interfering RNAs (siRNAs). siRNAs have an acceptable half-life in vitro, a predictable biodistribution profile similar to that of single-stranded antisense oligonucleotides (ASOs), and have repeatedly been more robust than ASO techniques in terms of consistency of transcript knockdown and threshold concentration. Following validation in mammalian cells by Tuschl and co-workers in 2001, synthetic siRNAs have gained wide acceptance as a laboratory tool for target validation. Currently, there is considerable interest in the therapeutic use of siRNA, particularly in areas of infectious disease and cancer. In vitro and in vivo findings demonstrate the efficacy of siRNA knockdown of gene messages that are pivotal for tumor cell growth, metastasis, angiogenesis and chemoresistance, leading to tumor growth suppression. However, siRNA-based cancer therapy faces similar pharmacokinetic limitations to ASO therapy with respect to the extent that siRNA accesses primary and metastatic target cells. The recently identified 'off-target activity' of siRNAs is also of concern. The concept of carrier-restricted delivery of siRNA by conditionally replicative, oncolytic adenoviruses is discussed. Oncolytic adenoviral delivery offers the potential benefits of restricted and renewable siRNA expression within the tumor microenvironment, an additive antitumor outcome through viral oncolysis and siRNA-mediated oncogene silencing, and a proven clinical platform with respect to infectivity and safety.

Adenoviridae↗
PubMed2002

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation.

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

Catalysis↗
PubMed1986

Deletion of the spf (spot 42 RNA) gene of Escherichia coli.

To investigate the function of spot 42 RNA, a small RNA of Escherichia coli, we constructed a strain in which spf, the structural gene for this RNA, is deleted. We achieved this by using a delta att phage lambda carrying a DNA fragment spanning the spf region but with a precise deletion of spf. By integration of this phage at the spf locus and by its subsequent excision, we were able to cross the spf deletion onto the bacterial chromosome. The fact that such a deletion could be obtained indicated that spf is not an essential gene. We did not observe any major defect in delta spf cells, although in one strain background the deletion caused a slight growth impairment.

Bacteriophage lambda↗
PubMed1989

The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth.

The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1). ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites [( rho-]) at high frequency, and showed signs of aberrant cell division. A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs.

Base Sequence↗
PubMed2006

Suppression of androgen receptor transactivation and prostate cancer cell growth by heterogeneous nuclear ribonucleoprotein A1 via interaction with androgen receptor coregulator ARA54.

The androgen receptor (AR) requires coregulators for its optimal transactivation. Whether AR coregulators also need interacting proteins to modulate their function remains unclear. Here we describe heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as an associated negative modulator for the AR coregulator ARA54. hnRNP A1 selectively suppressed ARA54-enhanced wild-type and mutant AR transactivation via interruption of AR-ARA54 interaction and ARA54 homodimerization. Stable transfection of hnRNP A1 in the LNCaP cells suppressed AR-mediated cell growth and the expression of prostate-specific antigen, and this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Small interfering RNA knockdown of endogenous hnRNP A1 enhanced cell growth and prostate-specific antigen expression in LNCaP cells. These results not only suggest that the loss of hnRNP A1 expression might activate the ARA54-enhanced cell growth and contribute to the prostate cancer progression, but also demonstrate the dual functional roles for ARA54 as an AR coregulator directly and as a mediator for the suppressive effect of hnRNP A1 indirectly. The novel finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach to battle prostate cancer by targeting AR indirectly with fewer side effects.

3T3 Cells↗
PubMed2003

Immune modulation by silencing IL-12 production in dendritic cells using small interfering RNA.

RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human and mouse. Specific gene silencing is mediated by short strands of duplex RNA of approximately 21 nt in length (termed small interfering RNA or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 production were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.

Adjuvants, Immunologic↗
PubMed1989

Stability of RNA chain elongation complexes formed with RNA polymerase and denatured DNA templates.

A random transcription system was used to analyze RNA chain-elongation complexes with RNA polymerases I and II from mouse ascites sarcoma cells. The molecular size of synthesized RNA was analyzed to characterize the elongation reaction by RNA polymerases. The RNA chain-elongation complex after initiation of RNA synthesis on denatured DNA by RNA polymerase II was not decomposed with heparin which dissociated the DNA-RNA polymerase complex, whereas the elongation complex on denatured DNA by RNA polymerase I was more susceptible to decomposition with heparin. RNA transcribed by RNA polymerase I on denatured DNA 30 min after the start of the reaction contained large RNA around 28S and small RNA. The small RNA could be formed by reinitiation and early termination of elongation. RNA transcribed by RNA polymerase II on denatured DNA was only large RNA around 28S. The elongation reaction by RNA polymerases I and II on denatured DNA was not inhibited by rifamycin AF/013, an inhibitor of initiation. These results suggest that RNA polymerase II could synthesize large transcripts without reinitiation and early termination of elongation and that RNA polymerase II formed more a stable elongation complex on denatured DNA than RNA polymerase I.

DNA↗
PubMed2002

Effective expression of small interfering RNA in human cells.

In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a double-stranded RNA (dsRNA) corresponding to exon sequences in the mRNA (refs 1,2). The use of this "RNA interference" (RNAi) in mammalian studies had lagged well behind its utility in lower animals because uninterrupted RNA duplexes longer than 30 base pairs trigger generalized cellular responses through activation of dsRNA-dependent protein kinases. Recently it was demonstrated that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (approximately 20 base pairs) through liposome transfection. We have explored several strategies for expressing similar short interfering RNA (siRNA) duplexes within cells from recombinant DNA constructs, because this might allow long-term target-gene suppression in cells, and potentially in whole organisms. Effective suppression of target gene product levels is achieved by using a human U6 small nuclear RNA (snRNA) promoter to drive nuclear expression of a single RNA transcript. The siRNA-like parts of the transcript consists of a 19 base pair siRNA stem with the two strands joined by a tightly structured loop and a U1-4 3' overhang at the end of the antisense strand. The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.

Base Sequence↗
PubMed1986

U6 small nuclear RNA is transcribed by RNA polymerase III.

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.

Amanitins↗
PubMed1998

Classification of gas5 as a multi-small-nucleolar-RNA (snoRNA) host gene and a member of the 5'-terminal oligopyrimidine gene family reveals common features of snoRNA host genes.

We have identified gas5 (growth arrest-specific transcript 5) as a non-protein-coding multiple small nucleolar RNA (snoRNA) host gene similar to UHG (U22 host gene). Encoded within the 11 introns of the mouse gas5 gene are nine (10 in human) box C/D snoRNAs predicted to function in the 2'-O-methylation of rRNA. The only regions of conservation between mouse and human gas5 genes are their snoRNAs and 5'-end sequences. Mapping the 5' end of the mouse gas5 transcript demonstrates that it possesses an oligopyrimidine tract characteristic of the 5'-terminal oligopyrimidine (5'TOP) class of genes. Arrest of cell growth or inhibition of translation by cycloheximide, pactamycin, or rapamycin-which specifically inhibits the translation of 5'TOP mRNAs-results in accumulation of the gas5 spliced RNA. Classification of gas5 as a 5'TOP gene provides an explanation for why it is a growth arrest specific transcript: while the spliced gas5 RNA is normally associated with ribosomes and rapidly degraded, during arrested cell growth it accumulates in mRNP particles, as has been reported for other 5'TOP messages. Strikingly, inspection of the 5'-end sequences of currently known snoRNA host gene transcripts reveals that they all exhibit features of the 5'TOP gene family.

3T3 Cells↗
PubMed1989

Nuclear factor I can functionally replace transcription factor Sp1 in a U2 small nuclear RNA gene enhancer.

Polymerase II transcription of a human gene for the small nuclear RNA U2 is dependent on two different promoter elements: a TATA-equivalent proximal sequence element and a distal enhancer element, which has been shown to contain Sp1- and octamer-binding sites. We have investigated the functional interplay between these transcription factor-binding sites of the enhancer, following transfection of U2 maxigene constructions into HeLa cells. There is a functional non-additive co-operation between the octamer-binding factor and Sp1, which is not dependent on the evolutionally conserved steric arrangement of these binding sites. We demonstrate that the conserved Sp1-binding site of the U2 enhancer can be fully substituted by a nuclear factor I (NFI) binding site, and that the octamer-binding factor functions in stimulating transcription in conjunction with either Sp1 or NFI. Since the octamer-binding factor is most probably the same protein as nuclear factor III (NFIII), the results imply that the NFI/NFIII complex, involved in adenovirus DNA replication, also can function as an efficient activator of transcription.

Base Sequence↗
PubMed2000

Phylogenetic relationships of human and wildlife piroplasm isolates in the western United States inferred from the 18S nuclear small subunit RNA gene.

The 18S nuclear small subunit ribosomal RNA gene of piroplasms from wildlife and human cases of babesiosis in the western USA were isolated by PCR and sequenced. Phylogenetic analyses of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that piroplasm isolates from the human cases were indistinguishable from some of the isolates from the western wildlife species, most notably the isolates from mule deer (Odocoileus hemionus). These results suggest that large ungulates may serve as reservoirs for human piroplasm infection. The western piroplasm isolates from humans and wildlife formed a distinct clade, separate from other piroplasms found worldwide.

Animals↗
PubMed2003

Lack of efficacy of 'naked' small interfering RNA applied directly to rat brain.

UNLABELLED: The intrastriatal infusions of 'naked' small interfering RNA (siRNA) targeted to dopamine D1 receptors (1.0-10.0 nmol over 3 days) did not reduce dopamine D1 receptor messenger RNA levels or receptor protein, assessed by [125I] SCH 23982 binding in intact rats. This was in contrast to results in vitro where a 76% reduction in dopamine D1 receptor ligand binding could be observed. CONCLUSION: The results suggest that synthetic siRNA, when applied directly to rat brain, is not capable of inducing RNA interference.

Animals↗