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PubMed1996

Characterization of the intron-encoded U19 RNA, a new mammalian small nucleolar RNA that is not associated with fibrillarin.

We have characterized a new member (U19) of a group of mammalian small nuclear RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date. Human U19 RNA is 200 nucleotides long and possesses 5'-monophosphate and 3'-hydroxyl termini. It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs. Human and mouse RNA are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA. In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts. During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of approximately 65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.

Animals↗
PubMed1995

Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

Base Sequence↗
PubMed1977

Comparative neutron small-angle scattering study of small spherical RNA viruses.

Small-angle neutron scattering from solutions of small RNA viruses has been used to study protein-nucleic acid organisation. In the five viruses investigated the RNA is confined to a sphere of about 100 A radius, with a central hole (with one possible exception). The interpenetration of RNA and protein varies with viruses and seems to be related to the natrue of the forces stabilising the virus.

Coliphages↗
PubMed1992

Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's.

Spliceosomal U6 small nuclear RNA (U6 RNA) in species as diverse as man, frog, fruitfly, and soybean have at their 3' ends a cyclic 2',3'-phosphate (greater than p) apparently derived from uridylic acid residues that were added post-transcriptionally. The 3' ends of U6 RNA's from various sources may be processed in different ways, or to different extents, depending on the organism or stage of development. The presence of a greater than p terminus on U6 RNA may influence the activity of U6 RNA either directly during splicing or indirectly by ensuring that the RNA has a defined length or proper conformation (or both).

Animals↗
PubMed2002

U87 RNA, a novel C/D box small nucleolar RNA from mammalian cells.

A novel 72 nt small nucleolar RNA (snoRNA) called U87 was found in rat liver cells. This RNA possesses the features of C/D box snoRNA family: boxes C, D', C', D, and 11 nt antisense element complementary to 28S ribosomal RNA (rRNA). The vast majority of C/D box snoRNAs direct site-specific 2'-O-ribose methylation of rRNAs. U87 RNA is suggested to be involved in 2'-O-methylation of a G(3468) residue in 28S rRNA. U87 RNA was detected in different mammalian species with slight length variability. Rat and mouse U87 RNA gene was characterized. Unlike the majority of C/D box snoRNAs U87 RNA lacks the terminal stem required for snoRNA processing. However, U87 gene is flanked by 7 bp inverted repeats potentially able to form a terminal stem in U87 RNA precursor.

Animals↗
PubMed1988

Transcription of a U6 small nuclear RNA gene in vitro. Transcription of a mouse U6 small nuclear RNA gene in vitro by RNA polymerase III is dependent on transcription factor(s) different from transcription factors IIIA, IIIB, and IIIC.

U6 small nuclear RNA (snRNA), an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. In this study, the optimal conditions for transcription of the U6 snRNA gene in vitro were found to be similar to conditions optimal for transcription of 5S RNA genes. To purify the trans-acting factors necessary for the transcription of the U6 RNA gene, HeLa cell extracts were fractionated on a DEAE-Sephadex column, and three fractions, designated DE-50, DE-175, and DE-500, were obtained by stepwise elution with 50, 175, and 500 mM ammonium sulfate, respectively. DE-175 fraction transcribed tRNA and 5S RNA genes but not a mouse U6 RNA gene. Complementation of the DE-175 fraction with the DE-50 fraction resulted in the transcription of the U6 RNA gene. Experiments in which the transcription factor (TFIIIA) was selectively inactivated indicated that TFIIIA is not required for the transcription of the U6 RNA gene. These results show that the U6 snRNA gene, although transcribed by RNA polymerase III, differs from tRNA and 5S RNA genes in that factors other than TFIIIA, -IIIB, and -IIIC are required for U6 gene transcription in vitro.

Cloning, Molecular↗
PubMed2003

A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA.

The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs. In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure. U3 snoRNA contains two conserved pairs of boxes, C'/D and B/C, which were both expected to bind the 15.5-kD/Snu13 protein. Only binding to the B/C motif was experimentally demonstrated. Here, by chemical probing of in vitro reconstituted RNA/protein complexes, we demonstrate the independent binding of the 15.5-kD/Snu13 protein to each of the two motifs. Due to a highly reduced stem I (1 bp), the K-turn structure is not formed in the naked B/C motif. However, gel-shift experiments revealed a higher affinity of Snu13p for the B/C motif, compared to the C'/D motif. A phylogenetic analysis of U3 snoRNA, coupled with an analysis of Snu13p affinity for variant yeast C'/D and B/C motifs, and a study of the functionality of a truncated yeast U3 snoRNA carrying base substitutions in the C'/D and B/C motifs, revealed that conservation of the identities of residues 2 and 3 in the B/C K-turn is more important for Snu13p binding and U3 snoRNA function, than conservation of the identities of corresponding residues in the C'/D K-turn. This suggests that binding of Snu13p to K-turns with a very short helix I imposes sequence constraints in the bulge. Altogether, the data demonstrate the strong importance of the binding of the 15.5-kD/Snu13 protein to the C'/D and B/C motifs for both U3 snoRNP assembly and activity.

Base Sequence↗
PubMed2004

Application of RNA interference to chicken embryos using small interfering RNA.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Animals↗
PubMed1997

Identification and characterization of a nuclease specific for the 3' end of the U6 small nuclear RNA.

The U6 small nuclear RNA is post-transcriptionally processed by the addition and removal of nontemplate uridylates (Us) at its 3' end prior to incorporation into the U4 x U6 small nuclear ribonucleoprotein complex. An enzyme responsible for removing Us from the U6 3' end has not been previously identified. Here we biochemically isolate and characterize an exonuclease activity from HeLa cells that removes template and nontemplate 3'-nucleotides specifically from U6 RNA. We also report the isolation of an inhibitor of this U6 nuclease. U6 nuclease rapidly removes the four terminal 3' Us found in the human U6 coding sequence in a magnesium-dependent manner. Mutagenesis studies on the U6 RNA define regions essential for processing. U6 nuclease recognizes specific sequences on both strands near the base of the major intramolecular stem loop of the U6 RNA. The preprocessed 3' Us form part of the base of the stem loop, but neither specific sequences nor secondary structure at the four terminal nucleotides are required to achieve processing.

Base Sequence↗
PubMed2004

Down-regulation of human deoxyuridine triphosphate nucleotidohydrolase (dUTPase) using small interfering RNA (siRNA).

A small interfering double stranded RNA molecule (siRNA, 21 bp) corresponding to a portion (nucleotides 337 to 357) of domain 3 of the human dUTPase was synthesized and used to determine whether it could down-regulate dUTPase activity in human cells. Transfection of the siRNA into HeLa and HT29 cells resulted in a 56 +/- 3.6% decrease in dUTPase activity, while transfection of SW620 cells resulted in a 27 +/- 6% decrease in dUTPase activity when compared to non-treated controls.

Amino Acid Motifs↗
PubMed2001

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

Adenosine Triphosphate↗
PubMed2000

Small evolutionarily conserved RNA, resembling C/D box small nucleolar RNA, is transcribed from PWCR1, a novel imprinted gene in the Prader-Willi deletion region, which Is highly expressed in brain.

Prader-Willi syndrome is a complex neurodevelopmental disorder caused by the inactivation or deletion of imprinted, paternally expressed genes in chromosome band 15q11.2. We report the identification and characterization of PWCR1, a novel imprinted gene within that region, and its mouse orthologue, Pwcr1, which was mapped to the conserved syntenic region on mouse chromosome 7. Expressed only from the paternal allele, both genes require the imprinting-center regulatory element for expression and are transcribed from the same strand. They are intronless and do not appear to encode a protein product. High human/mouse sequence similarity (87% identity) is limited to a 99-bp region called "HMCR" (for "human-mouse conserved region"). The HMCR sequence has features of a C/D box small nucleolar RNA (snoRNA) and is represented in an abundant small transcript in both species. Located in nucleoli, snoRNAs serve as methylation guidance RNAs in the modification of ribosomal RNA and other small nuclear RNAs. In addition to the nonpolyadenylated small RNAs, larger polyadenylated PWCR1 transcripts are found in most human tissues, whereas expression of any Pwcr1 RNAs is limited to mouse brain. Genomic sequence analysis reveals the presence of multiple copies of PWCR1 and Pwcr1 that are organized within local tandem-repeat clusters. On a multispecies Southern blot, hybridization to an HMCR probe encoding the putative snoRNA is limited to mammals.

Alleles↗
PubMed1990

Anti-(U1) small nuclear RNA antibodies in anti-small nuclear ribonucleoprotein sera from patients with connective tissue diseases.

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. Sera from patients with connective tissue diseases often contain antibodies against the proteins present in these snRNPs. Antibodies against the RNA components of snRNPs, the U snRNAs, are thought to be rare. We tested 118 anti-snRNP sera for the presence of anti-snRNA antibodies and found them in 45 sera (38%). In all sera the antibodies (IgG and F(ab)2 fragments thereof) were exclusively directed against U1 snRNA. The anti-(U1) RNA antibodies were always accompanied by anti-(U1)RNP antibodies but were not found in sera which contain antibodies of the Sm serotype directed against all nucleoplasmic U snRNP particles. Like anti-RNP antibodies, anti-U1 RNA activity is confined to sera from patients with SLE or SLE overlap syndromes and is rarely found in patients with other connective tissue diseases. By analyzing binding to subfragments of U1 snRNA made in vitro, it was demonstrated that anti-(U1)RNA antibodies recognize epitopes distributed throughout the U1 RNA molecule. In most sera, however, either the second or the fourth hairpin loop is the main target of the antibody. The possible mechanisms that could lead to the production of this new type of autoantibody are discussed.

Antibody Specificity↗
PubMed2005

Stable triplet of uracil-uracil basepairs in a small antisense RNA.

The interaction between ant (antirepressor) mRNA and its antisense RNA, sar (small antisense RNA), is important in regulation of the development of bacteriophage P22. Sar is 68-69 nucleotides in length and is believed to consist of two hairpin structures separated by a small inter-hairpin region, followed by a short 3' tail [Schaefer and McClure RNA 1997, 3, 141-156]. A novel feature of the proposed secondary structure of the first hairpin is a extremely rare triple U:U base stack. Heteronuclear NMR studies presented here show that the first hairpin does not possess a unique structure in the absence of the second hairpin. However, it acquires a well-defined structure within full length sar. Remarkably, the triple U:U stack appears to be a stable feature of the first hairpin, regardless of the presence or absence of the second hairpin.

Base Pairing↗
PubMed1981

Novel models for RNA splicing that involve a small nuclear RNA.

Nucleotide sequences of mammalian small nuclear RNAs (snRNAs) have been analyzed with a computer program for complementarity with sequences around a splice junction of various eukaryotic mRNA precursors (pre-mRNAs). A region in U2 RNA or some other snRNAs can form base pairs with both exons surrounding an intron of certain pre-mRNAs and, thereby, can align the two junctions leading to correct splicing of the pre-mRNA. These findings suggest that a snRNA such as U2 can be involved in splicing certain pre-mRNAs by pairing with exons, which we we call an "exon model" for splicing, as compared with the model involving U1 RNA presented by Lerner et al. [Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin, S. L. & Steitz, J. A. (1980) Nature (London) 283, 220-224]. We constructed a secondary structure model of U1 RNA and studied the capacity of base pairing with pre-mRNAs on the basis of both primary and secondary structures of U1 RNA. We present an alternative model for splicing that involves U1 RNA, which assumes base pairing of noncontiguous regions of U1 RNA with an intron of a pre-mRNA. Pairing of an snRNA with exons could explain correct matching of the two junctions that bound one and the same intron, which is not explained by pairing with consensus sequences at the ends of an intron as proposed by Lerner et al. Pairing of an intron with U1 RNA and pairing of the surrounding exons with another snRNA such as U2 RNA could take place at the same time to insure specificity of splicing.

Animals↗
PubMed2002

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis.

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.

Apoptosis↗
PubMed2004

Transgene overexpression with cognate small interfering RNA in tobacco.

Small interfering RNAs (siRNAs) are a key component of RNA silencing, including cosuppression. Here, we show an example in which siRNA does not serve in the downregulation of target genes. A tobacco endoplasmic reticulum omega-3 fatty acid desaturase (NtFAD3) catalyzes the formation of alpha-linolenate (18:3). Introduction of the NtFAD3 gene into tobacco plants caused strong reduction of 18:3 content in leaf tissues, which is associated with the production of the NtFAD3 siRNAs. However, this silencing effect was lacking in the root tissues. Both the introduced NtFAD3 and endogenous NtFAD3 genes were expressed successfully, and the roots showed increased 18:3 phenotype. Surprisingly, the NtFAD3 siRNAs were produced even in the root tissues. Expression of a hairpin double-stranded RNA against the NtFAD3 gene caused efficient reduction of 18:3 content in root tissues. Therefore, cosuppression of the NtFAD3 gene in tobacco appears to include an as yet unidentified developmental stage and tissue-specific mechanism of regulation of siRNA function.

Fatty Acid Desaturases↗