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PubMed1983

Sequential synthesis of small capped RNA transcripts in vitro by vesicular stomatitis virus.

Using purified viral or intracellular transcriptive complexes (RNP cores) of vesicular stomatitis virus (VSV), we have identified several small RNA species, ranging in size from 12 to 47 nucleotides in length that are synthesized in vitro by the genomic RNA. One group of small RNA transcripts is composed of three species that are capped at their 5' termini. Two of the capped species are from the start of the N gene and one is from the start of the NS gene. Unlike the previously described 5' triphosphated small RNAs, the templates encoding these small capped RNAs had uv target sizes greater than their respective lengths. In addition, these RNAs appeared sequentially during synchronized in vitro transcription reactions. Thus, these results provide evidence that sequences representing the 5'-capped termini of N and NS mRNAs are synthesized concomitantly with their respective mRNAs rather than simultaneously at the onset of transcription as proposed for the multiple entry, start-stop model (D. Testa, P. K. Chanda, and A. K. Banerjee, 1980, Cell 21, pp. 267-275). Together with the inability of the internally initiated 5'-triphosphated RNAs to be chased into mRNA (R. A. Lazzarini, I. Chien, F. Yang, and J. D. Keene, 1982, J. Gen. Virol. 58, 429-441), these results support a single entry model of VSV mRNA transcription.

Genes, Viral↗
PubMed2005

DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana.

Arabidopsis thaliana contains four DICER-LIKE (DCL) genes with specialized functions in small RNA biogenesis for RNA interference-related processes. A mutant with defects in DCL4 was identified and analyzed for microRNA- and endogenous, small interfering RNA (siRNA)-related functions. The dcl4-2 mutant contained normal or near-normal levels of microRNAs (21 nt) and heterochromatin-associated siRNAs (24 nt). In contrast, this mutant lacked each of three families of 21-nt trans-acting siRNAs (ta-siRNAs) and possessed elevated levels of ta-siRNA target transcripts. The dcl4-2 mutant resembled an rna-dependent RNA polymerase 6 mutant in that both mutants lacked ta-siRNAs and displayed heterochronic defects in which vegetative phase change was accelerated. Double mutant analyses with dcl2-1, dcl3-1, and dcl4-2 alleles revealed hierarchical redundancy among DCL activities, leading to alternative processing of ta-siRNA precursors in the absence of DCL4. These data support the concept that plants have specialized and compartmentalized DCL functions for biogenesis of distinct small RNA classes.

Arabidopsis↗
PubMed1996

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Amino Acid Sequence↗
PubMed2006

Gene-expressed RNA as a therapeutic: issues to consider, using ribozymes and small hairpin RNA as specific examples.

In recent years there has been a greater appreciation of both the role of RNA in intracellular gene regulation and the potential to use RNA in therapeutic modalities. In the latter case, RNA can be used as a therapeutic target or a drug. The chapters in this volume cover the varied and potent actions of RNA as antisense, ribozymes, aptamers, microRNA and small hairpin RNA in gene regulation, as well as their use as potential therapeutics for metabolic and infectious diseases. Our group has been involved in the development of anti-HIV gene expression constructs to treat HIV. In this chapter, we address the relevant scientific and some of the commercial issues in the use of RNA as a therapeutic. Specifically, the chapter discusses delivery, expression, potency, toxicity and commercial development using, as examples, hammerhead ribozymes and small hairpin RNA.

Animals↗
PubMed2005

Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library.

We have developed an original vector library that allowed us to exploit the phenomenon of RNA interference but also allowed us to avoid the confounding effects of the interferon response. In the present work, we used our library of small interfering RNA expression vectors to examine the genes involved in apoptosis that was induced by double-stranded RNA. To our surprise, screening of our library revealed two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. We also found that MST2 and protein kinase Calpha both activated the pro-apoptotic signal mediated by ERK2. The results of our screening analysis suggested the utility of large scale screenings with libraries of small interfering RNA expression vectors.

Apoptosis↗
PubMed2006

Analysis of Affymetrix GeneChip data using amplified RNA.

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.

Algorithms↗
PubMed2004

Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

Arabidopsis↗
PubMed1994

m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain.

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.

Animals↗
PubMed1986

Occurrence of novel small RNAs with concomitant inhibition of host cellular U small nuclear RNA synthesis in Vero cells infected with herpes simplex virus type 1.

In eukaryotic cells, small nuclear and small cytoplasmic RNAs (sn- or scRNAs) are associated with distinct proteins, forming ribonucleoproteins (snRNPs or scRNPs). In the present study we analysed the protein composition as well as the small RNA pattern in non-infected and herpes simplex virus type 1 (HSV)-infected Vero cells. We found that concomitantly with the shut-off of host cell mRNA synthesis, synthesis of U-snRNAs was stopped. Due to their stability, however, U-snRNAs were still present in cells 36 h after HSV infection. Besides these RNAs, two novel small RNAs which we termed HVR1 and HVR2 were detected in infected cells. On the basis of their relative mobilities in urea gels, the apparent chain lengths of these newly synthesized RNAs were determined to be 255 and 154 nucleotides respectively. The small RNA-binding proteins Sm, RNP, Ro and La were found to increase up to 15-fold after HSV infection. The data presented suggest that new, virus-coded small RNPs are synthesized which might play a role in the maturation and regulation of HSV-coded RNA transcripts.

Animals↗
PubMed1976

Particular small size RNA and RNA fragments from different origins as tumor inducing agents in Datura stramonium.

Particular RNA fragments obtained by action of pancreatic ribonuclease on purified RNAs originating from species totally unrelated to Agrobacterium tumefaciens (Escherichia coli, rabbit, monkey) are capable of inducing the formation of transplantable tumorous tissue when introduced at wounded sites in inverted stems of Datura stramonium maintained under axenic conditions on a medium containing auxin and kinetin. Reovirus RNA and a small size RNA (5-6S) isolated from RNA bound RNA directed DNA polymerase from Escherichia coli also induced the appearance of tumorous tissues which grow on solid synthetic medium in the absence of auxin and kinetin.

Animals↗
PubMed1994

RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.

Transcription of U2 small nuclear RNA (snRNA) genes in eukaryotes is executed by RNA polymerase II and is dependent on extragenic cis-acting regulatory sequences which are not found in other genes. Here we have mapped promoter elements of the Trypanosoma brucei U2 snRNA gene by transient DNA expression of mutant constructs in insect form trypanosomes. Unlike other eukaryotic U2 snRNA genes, the T. brucei homolog is transcribed by an RNA polymerase III-like enzyme on the basis of its sensitivity to the inhibitors alpha-amanitin and tagetitoxin. Thus, the trypanosome U2 snRNA provides a unique example of an RNA polymerase III transcript carrying a trimethylated cap structure. The promoter of this gene consists of three distinct elements: an intragenic sequence close to the 5' end of the coding region, which is probably required to position the polymerase at the correct transcription start site; and two extragenic elements, located 110 and 160 nucleotides upstream, which are essential for U2 snRNA gene expression. These two elements closely resemble both in sequence and in distance from each other the A and B box consensus sequences of the internal control regions of tRNA genes.

Amanitins↗
PubMed2005

UV-crosslinking of E1 small nucleolar RNA to proteins in frog oocytes.

E1/U17 small nucleolar RNA (snoRNA) is a box H/ACA snoRNA. To detect protein bands that UV-crosslink to E1 RNA primarily at uridines, frog oocytes were injected with [alpha-32P]UTP-labeled E1 RNA and incubated, isolated nuclei were UV irradiated, and nuclear contents were digested with RNase A. Wild-type E1 RNA specifically UV-crosslinked to several protein bands. To identify E1 RNA sites involved in these interactions, we tested 21 E1 RNA mutants, each consisting of substitutions in a conserved sequence or structure. UV-crosslinking of different protein bands to E1 RNA depended on one of the following sets of conserved E1 RNA segments: two 5' end RNA sites; five 5' half RNA sites; two 3' half RNA sites; or 14 sites located throughout E1 RNA. Of these conserved E1 RNA sites, UV-crosslinking apparently depended on sequences at 11 sites, and structures at 2 sites. Gel electrophoresis with and without RNA competition detected protein bands that are not common to all of the box H/ACA snoRNAs.

Animals↗
PubMed2003

Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA.

OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. METHODS: Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells. RESULTS: Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. CONCLUSION: siRNA may be effective in inhibiting SARS-associated coronavirus replication.

Animals↗
PubMed1986

Isolation and characterization of a human U3 small nucleolar RNA gene.

U3 RNA is an abundant, capped, small nucleolar RNA, implicated in the processing of preribosomal RNA. In this study, a DNA clone coding for U3 RNA (clone U3-1) was isolated from a human genomic library and characterized. The DNA sequence was identical to that of human U3 RNA isolated from HeLa cells. The flanking regions showed homology to the enhancer, promoter, and 3'-processing signal found in U1 and U2 snRNA genes. Further, the recently identified "U3 box" (GATTGGCTGCN10TATGTTAATTATGG) of rat U3 genes (Stroke and Weiner, (1985) J. Mol. Biol. 184, 183-193), was also found in the human U3 gene. This gene was transcribed in Xenopus oocytes; it is the first cloned true human U3 gene.

Base Sequence↗
PubMed1992

Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes.

The promoters of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes contain an essential and highly conserved proximal sequence element (PSE) approximately 55 bp upstream from the transcription start site. In addition, the upstream enhancers of all snRNA genes contain binding sites for octamer-binding transcription factors (Octs), and functional studies have indicated that the PSE and octamer elements work cooperatively. The present study has identified and characterized a novel transcription factor (designated PTF) which specifically binds to the PSE sequence of both RNA polymerase II- and RNA polymerase III-transcribed snRNA genes. PTF binding is markedly potentiated by Oct binding to an adjacent octamer site. This potentiation is effected by Oct-1, Oct-2, or the conserved POU domain of these factors. In agreement with these results and despite the independent binding of Octs to the promoter, PTF and Oct-1 enhance transcription from the 7SK promoter in an interdependent manner. Moreover, the POU domain of Oct-1 is sufficient for significant in vitro activity in the presence of PTF. These results suggest that essential activation domains reside in PTF and that the potentiation of PTF binding by Octs plays a key role in the function of octamer-containing snRNA gene enhancers.

Base Sequence↗
PubMed1994

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

Arabidopsis↗
PubMed1996

Cloning of two proximal sequence element-binding transcription factor subunits (gamma and delta) that are required for transcription of small nuclear RNA genes by RNA polymerases II and III and interact with the TATA-binding protein.

The proximal sequence element (PSE)-binding transcription factor (PTF) specifically recognizes the PSEs of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes. We previously have shown that PTF purified from human HeLa cells is a multisubunit complex of four polypeptides designated PTF alpha, -beta, -gamma, and -delta. We now report the isolation and expression of cDNAs encoding PTF gamma and PTF delta, as well as functional studies with cognate antibodies that recognize the native PTF complex in HeLa extracts. Immunoprecipitation studies confirm that the four PTF subunits originally found to copurify during conventional chromatography indeed form a tightly associated complex; they further show that the PTF so defined, including the gamma and delta subunits specifically, is essential for transcription of both class II and class III snRNA genes. Immunoprecipitation assays also show a weak substoichiometric association of the TATA-binding protein (TBP) with PTF, consistent with the previous report of a PTF-related complex (SNAPc) containing substoichiometric levels of TBP and a component (SNAPc43) identical in sequence to the PTF gamma reported here. Glutathione S-transferase pulldown assays further indicate relatively strong direct interactions of both recombinant PTF gamma and PTF delta with TBP, consistent either with the natural association of TBP with PTF in a semistable TBP-TBP-associated factor complex or with possible functional interactions between PSE-bound PTF and TATA-bound TBP during promoter activation. In addition, we show that in extracts depleted of TBP and TBP-associated factors, transcription from the U1 promoter is restored by recombinant TBP but not by TFIID or TFIIIB, indicating that transcription of class II snRNA genes requires a TBP complex different from the one used for mRNA-encoding genes.

Amino Acid Sequence↗
PubMed2000

Evolutionary conservation of post-transcriptional 3' end adenylation of small RNAs: S. cerevisiae signal recognition particle RNA and U2 small nuclear RNA are post-transcriptionally adenylated.

The 3' terminal nucleotide of several human small RNAs, including Signal Recognition Particle (SRP) RNA, 7SK RNA, U2 small nuclear RNA and ribosomal 5S RNA was previously characterized and a fraction of these RNAs was found to contain a single post-transcriptionally added adenylic acid residue on their 3' ends. Here we report the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for determining and quantifying the extent of post-transcriptional adenylation of RNAs from different species. Using this assay, we found that a fraction of S. cerevisiae U2 small nuclear RNA and S. cerevisiae SRP RNA contain a post-transcriptionally added adenylic acid residue on their 3' ends. Sequencing analysis confirmed this adenylation to be post-transcriptional. Corresponding small RNAs in Xenopus oocytes also contained this post-transcriptional adenylation on their 3' ends. These data show that post-transcriptional adenylation on the 3' end of several small RNA molecules is conserved through evolution. Xenopus SRP RNA from both cytoplasmic and nuclear compartments contained post-transcriptionally added adenylic acid residue on its 3' end. In addition, the Alu portion of SRP RNA was adenylated, when injected into the cytoplasm of frog oocytes. These data show that this novel adenylating machinery, capable of specifically adding a single adenylic acid to the 3' end of some RNA molecules, is present and functional in both nucleus and cytoplasm.

Adenosine Monophosphate↗