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PubMed2006

Argonaute slicing is required for heterochromatic silencing and spreading.

Small interfering RNA (siRNA) guides dimethylation of histone H3 lysine-9 (H3K9me2) via the Argonaute and RNA-dependent RNA polymerase complexes, as well as base-pairing with either RNA or DNA. We show that Argonaute requires the conserved aspartate-aspartate-histidine motif for heterochromatic silencing and for ribonuclease H-like cleavage (slicing) of target messages complementary to siRNA. In the fission yeast Schizosaccharomyces pombe, heterochromatic repeats are transcribed by polymerase II. We show that H3K9me2 spreads into silent reporter genes when they are embedded within these transcripts and that spreading requires read-through transcription, as well as slicing by Argonaute. Thus, siRNA guides histone modification by basepairing interactions with RNA.

Amino Acid Motifs↗
PubMed2004

Immunopurification and analysis of protein and RNA components of mRNP in mammalian cells.

We describe a basic, fast, and reliable technique to isolate and characterize ribonucleoprotein (RNP) using antibody to a constituent protein. The antibody serves to immunopurify RNP from total cells or nuclear and cytoplasmic cell fractions under conditions that promote RNP integrity. The presence of other RNP proteins as well as transcripts can then be analyzed by Western blotting and reverse transcription polymerase chain reaction, respectively. RNase treatment before immunopurification can be used to assess the dependence of protein-protein interactions on RNA. We also describe a modification using beta-mercaptoethanol that facilitates analyzing proteins that comigrate with antibody light or heavy chains.

Animals↗
PubMed1981

Structure-function relationship in Escherichia coli initiation factors. Identification of a lysine residue in the ribosomal binding site of initiation factor by site-specific chemical modification with pyridoxal phosphate.

Incubation of Escherichia coli initiation factor 3 (IF3) with pyridoxal phosphate (PLP) followed by reduction with sodium borohydride resulted in the selective modification and inactivation of this protein. The ribosomal-binding site (RNA-binding site) of IF3 is the target of PLP modification, since (a) the phosphate residue of PLP is required for inactivation; (b) RNA as well as synthetic polynucleotides (especially guanine-containing one) protect IF3 from inactivation; and (c) 30 S, but not 50 S ribosomal subunits, protect IF3 from PLP modification and from inactivation. The incorporation of PLP into IF3 occurred exclusively at lysine residues by reduction of the Schiff bases yielding epsilon-(5'-phosphopyridoxyl)lysine. The PLP-modified lysines were identified by amino acid analysis and sequencing of the PLP-modified peptides. Out of the 20 lysines of the factor, only Lys 2, Lys 5, Lys 99, Lys 112, Lys 166, and an unidentified Lys of the central cluster of the molecule (Lys 86, 87, 90, 91, 96) were found to be modified to varying degrees. The incorporation of 3 to 4 mol of PLP/mol of IF3 is accompanied by a substantial (greater than or equal to 80%) inactivation of this protein; the loss of activity follows apparent first order kinetics, and the inactivation results from the modification of just 1 Lys residue. This essential Lys residue was identified by various criteria to be Lys 112. The identification of an "active region" in the IF3 molecule is emerging from this as well as from other chemical modification studies.

Amino Acids↗
PubMed1993

2'-O-methyl RNA oligonucleotides identify two functional elements in the trypanosome spliced leader ribonucleoprotein particle.

Using permeable trypanosomes as an in vivo model system for trans-splicing, we have searched for functional elements in the Trypanosoma brucei spliced leader (SL) RNA by masking various regions of the molecule with short antisense 2'-O-methyl RNA oligomers. Initial probing of the structure of newly synthesized SL RNA by deoxyoligonucleotide-directed ribonuclease (RNase) H cleavage revealed three accessible regions: the 5' end, sequences downstream of the 5' splice site, and a putative single-stranded sequence between stem-loops II and III, which is thought to be analogous to the mammalian Sm-binding site of U small nuclear RNAs. Using antisense 2'-O-methyl RNA oligomers, two functional elements of the SL RNA became apparent. Masking of positions 1-18 inhibited modification of the cap 4 structure of newly synthesized SL RNA and, thereby, blocked utilization of the SL RNA in trans-splicing. In addition, nucleotides +1 to +4 relative to the 5' splice site, which include the invariant GU dinucleotide were accessible to oligomer binding in the SL ribonucleoprotein particle, and their blockade resulted in complete inhibition of trans-splicing. In contrast, RNA oligomer binding to the single-stranded region between stem-loop II and III of the SL RNA had no detectable effect on trans-splicing activity of the SL RNA.

Animals↗
PubMed1999

RNA editing of a Drosophila sodium channel gene.

Extensive analysis of cDNAs from the para locus in D. melanogaster reveals posttranscriptional modifications indicative of adenosine-to-inosine RNA editing. Most of these edits occur in highly conserved regions of the Na+ channel, and they occur in distant relatives of D. melanogaster as well. Sequence comparison between species has identified putative cis-acting elements important for each RNA editing site. Double-stranded RNA secondary structures with striking similarity to known RNA editing sites were generated based on these data. In addition, the RNA editing sites appear to be developmentally regulated. We have cloned a potential RNA editase, DRED, with a high degree of homology to the mammalian RED1,2 genes. The DRED locus itself is highly regulated by transcription from alternative promoters and alternative splicings.

Adenosine Deaminase↗
PubMed2001

Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses.

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.

Anticodon↗
PubMed1997

Inhibition of Hepatitis Delta Virus Genomic Ribozyme Self-Cleavage by Aminoglycosides.

Subgenomic regions of hepatitis delta virus (HDV) RNA contains ribozyme whose activities are important to viral life cycles and depend on a unique pseudoknot structure. To explore the characters of HDV ribozyme, antibiotics of the aminoglycoside, which has been shown inhibiting self-splicing of group I intron and useful in elucidating its structure, were tested for their effect on HDV genomic ribozyme. Aminoglycosides, including tobramycin, netromycin, neomycin and gentamicin effectively inhibited HDV genomic ribozyme self-cleavage in vitro at a concentration comparable to that inhibiting group I intron self-splicing. The extent of inhibition depended upon the concentration of magnesium ion. Chemical modification mapping of HDV ribozyme RNA indicated that the susceptibility of nucleotide 703 to the modifying agent was enhanced in the presence of tobramycin, suggesting a conformational shift of HDV ribozyme, probably due to an interaction with the aminoglycoside. Finally, we examined the effect of aminoglycoside on HDV cleavage and replication in cell lines, however, none of the aminoglycoside effective in vitro exerted suppressive effects in vivo. Our results represented as an initial effort in utilizing aminoglycoside to probe the structure of HDV ribozyme and to compare its reaction mechanism with those of other related ribozymes.

Journal Article↗
PubMed2003

The small RNA profile during Drosophila melanogaster development.

Small RNAs ranging in size between 20 and 30 nucleotides are involved in different types of regulation of gene expression including mRNA degradation, translational repression, and chromatin modification. Here we describe the small RNA profile of Drosophila melanogaster as a function of development. We have cloned and sequenced over 4000 small RNAs, 560 of which have the characteristics of RNase III cleavage products. A nonredundant set of 62 miRNAs was identified. We also isolated 178 repeat-associated small interfering RNAs (rasiRNAs), which are cognate to transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs participate in defining chromatin structure. rasiRNAs are most abundant in testes and early embryos, where regulation of transposon activity is critical and dramatic changes in heterochromatin structure occur.

Animals↗
PubMed1993

Modification of a free Fe-S cluster cysteine residue in the active iron-responsive element-binding protein prevents RNA binding.

The iron-responsive element-binding protein (IRE-BP) binds to specific RNA stem-loop structures called iron-responsive elements (IREs), which mediate the post-transcriptional regulation of a variety of genes involved in iron metabolism. The IRE-BP is cytosolic aconitase, and a [4Fe-4S] cubane cluster is required for aconitase activity but is associated with loss of IRE binding affinity. Chemical modification of the IRE-BP can abrogate RNA binding and the 3 cysteines predicted to coordinate the Fe-S cluster in the IRE-BP could be targets for modification. We report the expression of recombinant IRE-BP in which the three putative cluster cysteines (Cys-437, Cys-503, and Cys-506) have been mutated to serine residues. Replacement of any or all of these cysteine residues results in a complete loss of aconitase activity. While all of the mutants bind RNA, substitution of Cys-437 specifically renders the IRE-BP resistant to inactivation by low concentrations of N-ethylmaleimide or diamide. These results identify Cys-437 as the target of in vitro regulation of RNA binding in the IRE-BP and suggest that, in the RNA-binding form of the protein, Cys-437 is free and therefore available for modifications that inhibit RNA binding.

Amino Acid Sequence↗
PubMed1999

Synthesis of infectious viroids and other circular RNAs.

Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact-length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules.

DNA, Complementary↗
PubMed1988

Quiescent LLC-PK1 cells as a model for cis-diamminedichloroplatinum(II) nephrotoxicity and modulation by thiol rescue agents.

Confluent LLC-PK1 cells express many characteristics of renal proximal tubule epithelia. We report here the use of this cell line to investigate the possible mechanisms of cis-diamminedichloroplatinum(II) (DDP)-induced nephrotoxicity and diethyldithiocarbamate (DDTC) amelioration. Cells were exposed to platinum-based drug for 1 h and then incubated in drug-free medium until assayed. There was a 10-h delay between DDP exposure and onset of toxicity which then continued to develop. Viability at 72 h was 97 +/- 2 (SD), 68 +/- 3, 33 +/- 3, and 10 +/- 2% of control after treatment with 100, 200, 300, and 400 microM DDP, respectively. trans-Diamminedichloroplatinum(II) was 5-fold less toxic than DDP and diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) was nontoxic at concentrations up to 2.0 mM. Incubation of cells with DDTC (3 mM) for 1 h immediately following DDP exposure resulted in chelation of 43% of total intracellular platinum by DDTC and significantly increased 72 h viability; 97 +/- 2, 88 +/- 3, and 42 +/- 2% of control for 200, 300, and 400 microM DDP, respectively. DDP treatment of L1210 cells yielded equivalent total intracellular platinum levels, but Pt(DDTC)2 concentrations were one-tenth those in LLC-PK1 cells after subsequent treatment with DDTC (3 mM). Immediate reduction of protein and RNA, but not DNA, synthesis by DDP was concentration dependent over the same range as viability. DDP-induced increases in unscheduled DNA synthesis also did not correlate with cytotoxicity. The inhibition of protein synthesis was unchanged by pretreatment with the RNA synthesis inhibitor actinomycin D.DDTC (3 mM) exposure produced an immediate and persistent DDP dose modification of 1.6 in protein but not RNA synthesis. CBDCA (0.5 to 1.0 mM) had no effect on protein, RNA, or DNA synthesis and only slight stimulatory effects on unscheduled DNA synthesis. DDTC alone (3-3000 microM) caused significant reduction in DNA synthesis and unscheduled DNA synthesis. Neither sodium thiosulfate nor 2-mercaptoethane-sulfonate had any effect on DDP-induced cytotoxicity or inhibition of protein, RNA, or DNA synthesis when incubated immediately after DDP, even though these drugs achieved intracellular concentrations at which DDTC was protective. These data indicate that quiescent LLC-PK1 cells are a good in vitro model for the study of DDP-induced nephrotoxicity and its modulation by thiol rescue agents and that DDP inhibition and DDTC rescue of posttranscriptional processes correlate best with viability in LLC-PK1 cells.

Adenine Nucleotides↗
PubMed2001

Interactions between 23S rRNA and tRNA in the ribosomal E site.

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.

Anticodon↗
PubMed2006

Effect of UTP sugar and base modifications on vaccinia virus early gene transcription.

Prior efforts demonstrated that RNA oligonucleotides containing the transcription termination signal UUUUUNU stimulate premature termination of vaccinia virus early gene transcription, in vitro. This observation suggests that viral transcription termination may be an attractive target for the development of anti-poxvirus agents. Since short RNA molecules are readily susceptible to nuclease digestion, their use would require stabilizing modifications. In order to evaluate the effect of both ribose and uracil modifications of the U5NU signal on early gene transcription termination, UTP derivatives harboring modifications to the uracil base, the 2' position of the ribose sugar and the phosphodiester bond were examined in an in vitro vaccinia virus early gene transcription termination system. Incorporation of 4-S-U, 5-methyl-U, 2-S-U, pseudo U and 2'-F-dU into the nascent transcript inhibited transcription termination. 6-aza-U, 2'-amino-U, 2'-azido-U and 2'-O methyl-U inhibited transcription elongation resulting in the accumulation of short transcripts. The majority of the short transcripts remained in the ternary complex and could be chased into full-length transcripts. Initially, derivatives of all uridines in the termination signal were tested. Partial modification of the termination signal reduced termination activity, as well. Introduction of 2'-O methyl ribose to the first three uridines of the U9 termination signal reduced the ability of U9 containing oligonucleotides to stimulate in vitro transcription termination, in trans. Further modifications eliminated this activity. Thus, viral early gene transcription termination demonstrates a rigorous requirement for a U5NU signal that is unable to tolerate modification to the base or sugar. Additionally, VTF was shown to enhance transcription elongation through the T9 sequence in the template. These results suggest that VTF may play a subtle role in early gene transcription elongation in addition to its known function in mRNA cap formation, early gene transcription termination and intermediate gene transcription initiation.

Animals↗
PubMed2002

DNA and RNA modified dendritic cell vaccines.

The purpose of this paper is to describe the strategies for genetic modification of dendritic cells for use in active immunotherapy for the treatment of malignancies. An accruing body of data supports the concept of directing a therapeutic immune response, mediated by the cellular arm of the immune system, against cancers in vivo. Dendritic cells are the most potent inducers of T cell-mediated immune responses against tumor-expressed antigens, by virtue of their ability to present tumor antigens in the context of major histocompatibility molecules to naïve T cells with receptors specific for the antigen. A variety of methods for directing antigens to the MHC molecules of dendritic cells and augmenting their T cell stimulatory activity have been developed. This paper describes one group of promising strategies, the genetic modification of dendritic cells to direct the expression of tumor antigens, costimulatory molecules, and stimulatory cytokines. Genetic modification can be effected by either DNA or RNA, and the genetic material may be delivered by vectors or by physical means. These approaches are now entering human clinical trials.

Animals↗
PubMed1975

NMR investigation of the effect of selective modifications in the anticodon loop on the conformation of yeast transfer RNA-Phe.

Changes in the 300 MHz proton NMR spectrum of yeast tRNA-Phe induced by the removal of the Y-base from the anticodon loop are reversed when the dye proflavine is incorporated in its place. These observations correlate with our earlier interpretation of the NMR data that removal of the Y-base causes a change in the conformation of the anticodon stem. Such changes in stem conformation may in part be responsible for the differences in the biochemical properties of tRNA-Phe, tRNA-PhePF and tRNA-Phe-Y.

Anticodon↗
PubMed2003

Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1.

RNA binding proteins often contain multiple arginine glycine repeats, a sequence that is frequently methylated by protein arginine methyltransferases. The role of this posttranslational modification in the life cycle of RNA binding proteins is not well understood. Herein, we report that Sam68, a heteronuclear ribonucleoprotein K homology domain containing RNA binding protein, associates with and is methylated in vivo by the protein arginine N-methyltransferase 1 (PRMT1). Sam68 contains asymmetrical dimethylarginines near its proline motif P3 as assessed by using a novel asymmetrical dimethylarginine-specific antibody and mass spectrometry. Deletion of the methylation sites and the use of methylase inhibitors resulted in Sam68 accumulation in the cytoplasm. Sam68 was also detected in the cytoplasm of PRMT1-deficient embryonic stem cells. Although the cellular function of Sam68 is unknown, it has been shown to export unspliced human immunodeficiency virus RNAs. Cells treated with methylase inhibitors prevented the ability of Sam68 to export unspliced human immunodeficiency virus RNAs. Other K homology domain RNA binding proteins, including SLM-1, SLM-2, QKI-5, GRP33, and heteronuclear ribonucleoprotein K were also methylated in vivo. These findings demonstrate that RNA binding proteins are in vivo substrates for PRMT1, and their methylation is essential for their proper localization and function.

Adaptor Proteins, Signal Transducing↗