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PubMed1985

Transcription boundaries of U1 small nuclear RNA.

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

Base Sequence↗
PubMed1994

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

Base Sequence↗
PubMed2004

RNA interference--small RNAs effectively fight viral hepatitis.

RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by double-stranded RNA that is homologous in sequence to the target gene. This unique phenomenon has been extensively investigated during the last few years not only in the context of its mechanism and its possible role in the regulation of gene expression and cell function, but also as a potential powerful tool for gene therapy. Targeting essential viral genes or oncogenic alleles are only some of the possible applications of RNAi in the field of gene-directed therapy. This review covers the potential use of RNAi against hepatitis B and hepatitis C viruses, the main pathogens causing chronic liver disease. The major milestones along the discovery of RNAi will also be covered.

Animals↗
PubMed1989

Molecular analysis of dicot and monocot small nuclear RNA populations.

Oligonucleotides directed against conserved small nuclear RNA (snRNA) sequences have been used to identify the individual U1, U2, U4, U5, and U6 snRNAs in dicot and monocot nuclei. The plant snRNA populations are significantly more heterogeneous than the mammalian or Saccharomyces cerevisiae snRNA populations. U6 snRNA exists as a single species of similar size in monocot and dicot nuclei. The abundance and molecular weights of the U1, U2, U4, and U5 snRNAs expressed in monocot and dicot nuclei are significantly different. Whereas most dicot nuclei contain one or two predominant forms of U2 snRNA and a small number of U4 snRNAs, monocot nuclei contain multiple forms of U2 snRNA ranging from 208 to 260 nucleotides and multiple forms of U4 snRNA from 159 to 176 nucleotides. Multiple forms of U1 and U5 snRNA exist in both plant groups. All prominent size variants of U1, U2, U4, and U5 snRNA identified in monocot nuclei can be immunoprecipitated with anti-trimethylguanosine antibody. We conclude that the sizes and number of snRNA molecules involved in intron excision differ considerably in dicot and monocot nuclei. In wheat nuclei, we have identified an additional U1-like RNA that is differentially expressed during development.

Base Sequence↗
PubMed1998

Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae.

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C' and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5' portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.

Base Sequence↗
PubMed1995

Mutational analysis of Saccharomyces cerevisiae U4 small nuclear RNA identifies functionally important domains.

U4 small nuclear RNA (snRNA) is essential for pre-mRNA splicing, although its role is not yet clear. On the basis of a model structure (C. Guthrie and B. Patterson, Annu. Rev. Genet. 22:387-419, 1988), the molecule can be thought of as having six domains: stem II, 5' stem-loop, stem I, central region, 3' stem-loop, and 3'-terminal region. We have carried out extensive mutagenesis of the yeast U4 snRNA gene (SNR14) and have obtained information on the effect of mutations at 105 of its 160 nucleotides. Fifteen critical residues in the U4 snRNA have been identified in four domains: stem II, the 5' stem-loop, stem I, and the 3'-terminal region. These domains have been shown previously to be insensitive to oligonucleotide-directed RNase H cleavage (Y. Xu, S. Petersen-Bjørn, and J. D. Friesen, Mol. Cell. Biol. 10:1217-1225, 1990), suggesting that they are involved in intra- or intermolecular interactions. Stem II, a region that base pairs with U6 snRNA, is the most sensitive to mutation of all U4 snRNA domains. In contrast, stem I is surprisingly insensitive to mutational change, which brings into question its role in base pairing with U6 snRNA. All mutations in the putative Sm site of U4 snRNA yield a lethal or conditional-lethal phenotype, indicating that this region is important functionally. Only two nucleotides in the 5' stem-loop are sensitive to mutation; most of this domain can tolerate point mutations or small deletions. The 3' stem-loop, while essential, is very tolerant of change. A large portion of the central domain can be removed or expanded with only minor effects on phenotype, suggesting that it has little function of its own. Analysis of conditional mutations in stem II and stem I indicates that although these single-base changes do not have a dramatic effect on U4 snRNA stability, they are defective in RNA splicing in vivo and in vitro, as well as in spliceosome assembly. These results are discussed in the context of current knowledge of the interactions involving U4 snRNA.

Base Sequence↗
PubMed2006

RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium.

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with t-RNA(phe) was studied using various biophysical techniques and the data was compared with the binding of the classical DNA intercalator, ethidium. The results of optical thermal melting, differential scanning calorimetry and circular dichroism characterized the native cloverleaf structure of t-RNA under the conditions of the study. The strong binding of the alkaloids and ethidium to t-RNA was revealed from the absorption and fluorescence studies. The salt dependence of the binding constants enabled the dissection of the binding free energy to electrostatic and non-electrostatic contributions. This analysis revealed a surprisingly large favourable component of the non-electrostatic contribution to the binding of these charged alkaloids and ethidium to t-RNA. Isothermal titration calorimetric studies revealed that the binding of both the alkaloids is driven by a moderately favourable enthalpy decrease and a moderately favourable entropy increase while that of ethidium is driven by a large favourable enthalpy decrease. Taken together, the results suggest that the binding of these alkaloid molecules on the t-RNA structure appears to be mostly by partial intercalation while ethidium intercalates to the t-RNA. These results reveal the molecular aspects on the interaction of these alkaloids to t-RNA.

Alkaloids↗
PubMed1996

Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III.

The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF.

Amino Acid Sequence↗
PubMed1996

The Oct-1 POU-specific domain can stimulate small nuclear RNA gene transcription by stabilizing the basal transcription complex SNAPc.

The RNA polymerase II and III human small nuclear RNA promoters have a common basal element, the proximal sequence element, which binds the TATA box-binding protein-containing complex SNAPc. They also contain an enhancer characterized by a highly conserved octamer sequence, which constitutes a binding site for the broadly expressed POU domain transcription factor Oct-1. The POU domain is a bipartite DNA-binding domain consisting of a POU-homeo (POUH) domain and a POU-specific (POUs) domain joined by a flexible linker. Here, we show that the Oct-1 POU domain but not the related Pit-1 POU domain can facilitate the binding of SNAPc to the proximal sequence element, and activate transcription. The effect is probably mediated by protein-protein contacts, and 1 of 30 amino acid differences between the Oct-1 and Pit-1 POUs domains is the key determinant for the differential interaction with SNAPc and the ability to activate transcription. These results show that a function that is the hallmark of activation domains, namely, recruitment of a basal transcription complex resulting in activation of transcription, can be performed by a DNA-binding domain. In this case, subtle changes between activator DNA-binding domains, as subtle as a single amino acid difference, can profoundly affect interaction with the basal transcription machinery.

Amino Acid Sequence↗
PubMed2001

Reconstitution of transcription from the human U6 small nuclear RNA promoter with eight recombinant polypeptides and a partially purified RNA polymerase III complex.

The human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAP(c), and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAP(c) and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB", a human homologue of the B" subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAP(c), recombinant TBP, recombinant hB", and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of U3 small nucleolar RNA from the early diverging protist, Euglena gracilis.

U3 small nucleolar RNA (snoRNA) has been isolated from Euglena gracilis, an early diverging protist, and its primary sequence determined. Although this 180-nucleotide-long RNA is considerably smaller than its homolog in vertebrate animals, it contains the conserved sequence blocks (boxes A, Ao, B, C and D) characteristic of U3 snoRNAs from other organisms. A secondary structure can be modelled that displays many of the salient features found in published core structures of vertebrate, yeast and trypanosome U3 snoRNAs. The functional significance of this proposed secondary structure is discussed in relation to the role E. gracilis U3 snoRNA may have in pre-rRNA processing in this organism. Multiple expressed species of E. gracilis U3 snoRNA were found to differ in nucleotide sequence at a number of positions; some of these differences alter pairing in the proposed secondary structure. Analysis of E. gracilis genomic DNA revealed a complex pattern of U3-hybridizing sequences that parallels the multiplicity of expressed species of U3 snoRNA revealed by transcript analysis.

Animals↗
PubMed1984

Splicing of Xenopus laevis ribosomal protein RNAs is inhibited in vivo by antisera to ribonucleoproteins containing U1 small nuclear RNA.

The activity of antisera against ribonucleoproteins containing U1 small nuclear RNA (Sm and RNP) has been analysed on pol II transcripts in an in vivo system. Xenopus laevis ribosomal protein gene transcripts are accumulated in the form of precursor RNA when either of the two kinds of antisera are injected into the germinal vesicles of X. laevis oocytes before the injection of purified L1 and L14 ribosomal protein genes. No effect on the accumulation of mature histone mRNA is detected when X. laevis histone genes are injected together with the RNP antiserum. These results strongly suggest that U1-RNP complexes play an essential role in intron removal in vivo.

Animals↗
PubMed1986

Genes for human U4 small nuclear RNA.

A study of human genes coding for U4 small nuclear RNA is presented. It is known from previous studies that mammalian cells contain three major U4 RNA species, designated U4A, U4B, and U4C (Krol and Branlant, 1981). A clone was isolated from a human DNA library which contained two transcriptionally active genes for U4 RNA. U4 transcription was sensitive to low concentrations of alpha-amanitin, inferring that U4 RNA is a product of RNA polymerase II or RNA polymerase II-like activity. One of the two genes contains a coding region which matches the sequence of U4C RNA perfectly. The coding region of the second gene resembles U4B RNA although there are two differences between the sequence of this gene and the U4B RNA sequence, suggesting that it may encode a minor, hitherto undetected U4 RNA species. The 5'-flanking regions of the two U4 genes contain several almost perfectly conserved sequence motifs. One is located between positions -50 and -60. This motif is present in equivalent positions in the two U4 genes as well as in human U1 and U2 genes. A second motif, which is 19 nucleotides (nt) long and centered around nt position -140, is present in the two U4 genes but absent from U2 RNA genes. A third highly conserved region, located between nt positions -210 and -250, is a putative enhancer element. It includes one copy of the so-called octanucleotide motif, previously identified as adjacent to the early SV40 promoter and immunoglobulin promoters. Another highly conserved sequence motif, CTCTGTGA, is located approximately one helical turn upstream from the octanucleotide motif in both U2 and U4 genes. The human genome appears to contain a family of U4 RNA genes comprising at least 100 copies.

Animals↗
PubMed1994

A complex that contains proteins binding to the PSE and TATA sites in a human U6 small nuclear RNA promoter.

The proximal promoter of a human U6 small nuclear RNA (snRNA)-encoding gene contains two separate elements, the proximal sequence element (PSE) and the TATA box. We investigated the interaction of the PSE- and TATA-binding proteins (PBP and TBP) with normal and mutant U6 proximal promoters using an electrophoretic mobility shift assay. We detected a complex containing both PBP and TBP bound to the wild-type U6 promoter. Efficient formation of the triple complex was dependent on the presence of the PSE and the TATA box on the template DNA. Mutant U6 promoters containing an increased spacing between the PSE and TATA box of 5 or 10 bp were impaired in the ability to form a complex that includes TBP. We infer from these results that PBP and TBP interact when their binding sites are properly positioned in a U6 gene promoter.

Base Sequence↗
PubMed2003

Cloning and characterization of the Drosophila U7 small nuclear RNA.

Base pairing between the 5' end of U7 small nuclear RNA (snRNA) and the histone downstream element (HDE) in replication-dependent histone pre-mRNAs is the key event in 3'-end processing that leads to generation of mature histone mRNAs. We have cloned the Drosophila U7 snRNA and demonstrated that it is required for histone pre-mRNA 3'-end processing in a Drosophila nuclear extract. The 71-nt Drosophila U7 snRNA is encoded by a single gene that is embedded in the direct orientation in an intron of the Eip63E gene. The U7 snRNA gene contains conserved promoter elements typical of other Drosophila snRNA genes, and the coding sequence is followed by a 3' box indicating that the Drosophila U7 snRNA gene is an independent transcription unit. Drosophila U7 snRNA contains a trimethyl-guanosine cap at the 5' end and a putative Sm-binding site similar to the unique Sm-binding site found in other U7 snRNAs. Drosophila U7 snRNA is approximately 10 nt longer than mammalian U7 snRNAs because of an extended 5' sequence and has only a limited potential to form a stem-loop structure near the 3' end. The extended 5' end of Drosophila U7 snRNA can base pair with the HDE in all five Drosophila histone pre-mRNAs. Blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically blocks processing of a Drosophila histone pre-mRNA. Changes in the HDE that abolish or decrease processing efficiency result in a reduced ability to recruit U7 snRNA to the pre-mRNA.

Animals↗
PubMed2005

Application of small interfering RNA (siRNA) for modulation of airway epithelial gene expression.

Small interfering RNA (siRNA) was developed as a novel tool to inhibit gene function in human disease. The aim of the present study was to modify the function of NF-kappaB in airway epithelial cells by application of siRNA. 1HAEo cells were transfected with siRNA directed to the p65 subunit of NF- kappaB (siRNA.p65). Application of siRNA.p65 caused decreased levels of p65 mRNA or protein after 72 hours, as determined by quantitative RT-PCR or Western blot analysis. The tumor necrosis factor- alpha (TNF-alpha)-induced release of interleukin-6 (IL-6) and IL-8 was significantly inhibited by the application of siRNA.p65. Well-differentiated primary cells were resistant to transfection with siRNA.p65. However, when undifferentiated primary cells were transfected, an effect of the siRNA could still be observed when the cells were differentiated in an air-liquid interface culture system. In conclusion, siRNA can be used to regulate the activity of NF-kappaB in airway epithelial cells.

Bronchi↗
PubMed1992

Characterization of antibodies against methyl-pppN cap structure: plant U3 small nucleolar RNA is recognized by these antibodies.

In eukaryotes, many small nuclear RNAs contain either a trimethylguanosine cap structure of a gamma-monomethyl (me) cap structure. Previously, we reported the characterization of anti-mepppG antibodies which recognize methyl-capped RNAs with G as the initiation nucleotide. We report here the preparation of antibodies against mepppN cap structure. Anti-mepppN antibodies recognized only mepppN from a mixture of mepppN and pppN and immunoprecipitated mepppA-capped U3 small nucleolar RNA from a mixture of cowpea cell RNAs. These anti-mepppN antibodies recognized methylated nucleoside triphosphates (mepppA, mepppC, mepppG and mepppU) with nearly equal efficiency; however, these antibodies did not recognize methyl phosphate or methylated mononucleotides. These antibodies will be useful in the identification and characterization of all methyl-capped RNAs no matter which is the initiation nucleotide.

Antibodies, Antinuclear↗
PubMed1995

Accurate and efficient N-6-adenosine methylation in spliceosomal U6 small nuclear RNA by HeLa cell extract in vitro.

Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modifications including a single m6A (N-6-methyladenosine) at position 43. This A-43 residue is critical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m6A in the corresponding position showing that this modification is evolutionarily conserved. In this study, we show that upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to m6A-43. This conversion was detectable as early as 3 min after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m6A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m6A formation; however, the integrity of the 3' stem-loop was necessary for efficient m6A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m6A methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA. The enzyme that catalyzes m6A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts.

Adenosine↗