Binding of actinomycin D to DNA revealed by DNase I footprinting

We have analyzed the specificity of the actinomycin D-DNA interaction. The 'footprint' method has been used in this investigation. It is shown that: (i) The presence of dinucleotide GC or GG is required for binding of a single drug molecule. (ii) The strong binding sites are encoded by tetranucleotide XGCY; where X not equal to G and Y not equal to C in accordance with RNA elongation hindrance sites [1]. (iii) There is a positive cooperativity in binding of actinomycin D with DNA.     

Selection of the 3′-splice site in group I introns

A model for selection of 3′-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3′-splice site. This tertiary pairing would bring the 3′-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3′-exon sequences and the ‘internal guide sequence’ near the 5′-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3′-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.     

A structural database for k-turn motifs in RNA

The kink-turn (k-turn) is a common structural motif in RNA that introduces a tight kink into the helical axis. k-turns play an important architectural role in RNA structures and serve as binding sites for a number of proteins. We have created a database of known and postulated k-turn sequences and three-dimensional (3D) structures, available via the internet. This site provides (1) a database of sequence and structure, as a resource for the RNA community, and (2) a tool to enable the manipulation and comparison of 3D structures where known.     

Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N--terminal RRMs of CELF1

CUGBP, Elav-like family member 1 (CELF1) is an RNA binding protein with important roles in the regulation of splicing, mRNA decay and translation. CELF1 contains three RNA recognition motifs (RRMs). We used gel retardation, gel filtration, isothermal titration calorimetry and NMR titration studies to investigate the recognition of RNA by the first two RRMs of CELF1. NMR shows that RRM1 is promiscuous in binding to both UGU and CUG repeat sequences with comparable chemical shift perturbations. In contrast, RRM2 shows greater selectivity for UGUU rather than CUG motifs. A construct (T187) containing both binding domains (RRM1 and RRM2) was systematically studied for interaction with tandem UGU RNA binding sites with different length linker sequences UGU(U)_xUGU where x = 1–7. A single U spacer results in interactions only with RRM1, demonstrating both steric constraints in accommodating both RRMs simultaneously at adjacent sites, and also subtle differences in binding affinities between RRMs. However, high affinity co-operative binding (K_d ~ 0.4 µM) is evident for RNA sequences with x = 2–4, but longer spacers (x ≥ 5) lead to a 10-fold reduction in affinity. Our analysis rationalizes the high affinity interaction of T187 with the 11mer GRE consensus regulatory sequence UGUUUGUUUGU and has significant consequences for the prediction of CELF1 binding sites.     

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2′-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.     

The Nop5-L7A-fibrillarin RNP complex and a novel box C/D containing sRNA of Halobacterium salinarum NRC-1

RNA 2′O-methylation is a frequent modification of rRNA and tRNA and supposed to influence RNA folding and stability. Ribonucleoprotein (RNP) complexes, containing the proteins Nop5, L7A, fibrillarin, and a box C/D sRNA, are guided for 2′O-methylation by interactions of their RNA component with their target RNA. In vitro complex assembly was analyzed for several thermophilic Archaea but in vivo studies are rare, even unavailable for halophilic Archaea. To analyze the putative box C/D RNP complex in the extremely halophilic Halobacterium salinarum NRC-1 we performed pull-down analysis and identified the proteins Nop5, L7A, and fibrillarin and the tRNA^Trp intron, as a typical box C/D sRNA of this RNP complex in vivo. We show for the first time a ribonucleolytic activity of the purified RNP complex proteins, as well as for the RNP complex containing pull-down fractions. Furthermore, we identified a novel RNA (OE4630R-3′sRNA) as part of the complex, containing the typical boxes C/D and C′/D′ sequence motifs and being twice as abundant as the tRNA^Trp intron.     

The mechanism of inhibition of bacteriophage T7 RNA synthesis by acridines, diacridines and actinomycin D.

A homologous series of diacridines, as well as 9-amino acridine, were assayed for their ability to interfere with the synthesis of RNA (bands U-VI) by bacteriophage T7 DNA-dependent RNA polymerase transcribing T7 DNA in vitro; their action was compared to that of actinomycin D. It was found that, in contrast to actinomycin D which inhibits chain elongation, the acridines tested inhibited chain initiation only; no evidence for inhibition of chain elongation was noted. No clear-cut differentiation between single and double intercalators on the mechanism of inhibition of RNA synthesis could be determined, except that the latter are more potent inhibitors. However, it appears that diacridines connected with a diethyldiamine and a butyldiamine chain are less inhibitory to the synthesis of the RNA of Bands III and IV. The results furthermore indicate that the estimation of the number average molecular weight alone, without identification of the product RNA, is a potentially misleading method of determining the mode of action of these drugs.     

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ~2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca²⁺ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.     

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg²⁺ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.     

An Escherichia coli system for assay of Flp site-specific recombination on substrate plasmids

We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the Flp site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40. pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying Flp-recognition targets (FRT) are transformed into BL-FLP, and the consequences of Flp-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect Flp-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used Flp substrate plasmid, pNEOβGAL (O'Gorman et al. (1991) Science 251, 1351–1355).     

RNA polymerase II acts as an RNA‐dependent RNA polymerase to extend and destabilize a non‐coding RNA

RNA polymerase II (Pol II) is a well‐characterized DNA‐dependent RNA polymerase, which has also been reported to have RNA‐dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non‐coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3′‐end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α‐amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3′‐end.     

Circular RNAs are abundant,conserved, and associated with ALU repeats

Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.     

Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes

It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)⁺ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267–270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA “masking” proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation. © 1995 Wiley-Liss, Inc.