RNA cleavage linked with ribosomal action

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.     

Enzymatic cleavage of RNA by RNA

The catalytic activity of E. coli RNase P, an enzyme essential for tRNA biosynthesis in vivo, resides in the RNA subunit of the enzyme. This RNA, which has all the properties of a classical enzyme, can cleave precursor tRNAs in vitro in the total absence of proteins.     

Enzymatic cleavage of RNA by RNA

The discovery and characterization of the catalytic RNA subunit of the enzyme ribonuclease P ofEscherichia coli is described.Nobel lecture given on December 8, 1989, by Professor Sidney Altman, and published in LES PRIX NOBEL 1989, printed in Sweden by Norstedts Tryckeri, Stockholm, Sweden, 1990, republished here with the permission of the Nobel Foundation, the copyright holder.     

Site-directed cleavage of RNA.

Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region. Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem. The method is proved to be generally applicable to RNA containing secondary structures.     

Hybridase cleavage of RNA. I. Oligonucleotide probes for regiospecific RNA cleavage]

New oligonucleotide probes for regiospecific cleavage of RNA molecules by hybridase (RNase H) are suggested. RNase H from E. coli is shown to site-specifically split eight phosphodiester bonds in RNA in the heteroduplex, formed by 5S rRNA and d(ACCACCGCGCT). The partial substitution of deoxycytidines in position 5, 6, 8, 10 of the probe by 2'-O-methylcytidines leads to unique (regiospecific) RNA cleavage between U25 and C26.     

Directed cleavage of ribopolynucleotides with nucleases restricted by multiple modification of substrate.

Simultaneous exhaustive modification of cytidine and uridine residues of rRNA with methoxyamine and sodium metabisulfite renders adjacent phosphodiester bonds resistant to pancreatic and T2 ribonucleases. Another method of T2 RNAase restriction is modification of cytidine with methoxyaminebisulfite followed by modification of guanosine residues with beta-ethoxy-alpha-ketobutyraldehyde. Mild alkaline treatment leads to demodification of uridine and guanosine residues leaving intact modified cytidine residues, thus providing a means of stepwise, directed cleavage of the polynucleotide. The series of combined cleavage procedures and methods of isolation of oligo(C), oligo(G) and oligopyrimidine tracts, as well as the procedure of selective cleavage at uridine residues elaborated in the course of the present studies may serve as a basis for more rational procedures of RNA sequencing.     

Comparison of rRNA cleavage by complementary 1,10-phenanthroline-Cu(II)- and EDTA-Fe(II)-derivatized oligonucleotides

The chemical nucleases 1,10-phenanthroline-Cu(II) and EDTA-Fe(II), have proven to be valuable tools for structural analysis of nucleic acids. Both have found applications in footprinting and directed proximity studies of DNA and RNA. Derivatives of each that provide for tethering to nucleic acid or protein are commercially available, allowing their widespread use for structural analysis of macromolecules. Although their applications are somewhat overlapping, differences in their cleavage mechanisms and chemical properties allow them to provide distinct and complementary structural information. The purpose of this study is to compare directly the cleavage patterns of tethered 1,10-phenanthroline-Cu(II) and EDTA-Fe(II) complexes within a similar experimental system. Here, the region surrounding nucleotide 1400 of 16S rRNA from Escherichia coli serves as a substrate for chemical cleavage directed by a derivatized complementary oligonucleotide. This region of rRNA is known to be involved in the decoding of mRNA during translation. The results of this study provide evidence in support of the mechanistic differences previously established for EDTA-Fe(II) and 1,10-phenathroline-Cu(II). The delocalized cleavage envelope produced by EDTA-Fe(II) cleavage suggests the involvement of a diffusible reactive species. On the other hand, rRNA cleavage induced by the tethered 1,10-phenanthroline-Cu(II) complex appears localized to the proximity of the chemical nuclease under normal conditions, although the production of an unknown diffusible species appears to occur during long reaction times.     

Synthetic metallonucleases for RNA cleavage

Synthetic metallonucleases are versatile metal ion catalysts that use multiple catalytic strategies for the cleavage of RNA. Recent work in the design of more active metallonucleases combines a single metal ion with functional groups that interact with RNA, including amino acid fragments or additional metal ions. Rate enhancements by multifunctional catalysts for cleavage of simple model substrates with good leaving groups are as high as 10(6) but somewhat lower (10(5)) for real RNA. However, cleavage of RNA substrates is complicated by different binding modes and steric interactions that can interfere with catalysis. Antisense oligonucleotides, peptides and small molecules that act as RNA recognition agents increase the strength of substrate binding, but not necessarily the catalytic rate constant. In general, catalytic strategies used by synthetic metallonucleases are probably not optimized. A better grasp of the mechanism of RNA cleavage by metal ions and more effort on positioning the metal ion complex with respect to the cleavage site may lead to improved catalysts.     

Hybridase cleavage of RNA. V. Complementary precision of cleavage]

The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.     

Site specific enzymatic cleavage of RNA.

The hybridization of a DNA oligonucleotide a specific tetramer or longer) will direct a cleavage by RNase H (EC 3.1.4.34) to a specific site in RNA. The resulting fragments can then be labeled at their 5' or 3' ends, purified, and sequenced directly. This procedure is demonstrated with two RNA molecules of known sequence: 5.8S rRNA from yeast (158 nucleotides) and satellite tobacco necrosis virus (STNV) RNA (1240 nucleotides).     

Directed acyclic graph kernels for structural RNA analysis

Background  

Recent discoveries of a large variety of important roles for non-coding RNAs (ncRNAs) have been reported by numerous researchers. In order to analyze ncRNAs by kernel methods including support vector machines, we propose stem kernels as an extension of string kernels for measuring the similarities between two RNA sequences from the viewpoint of secondary structures. However, applying stem kernels directly to large data sets of ncRNAs is impractical due to their computational complexity.     

COS-7细胞中小发卡RNA介导的RNA干涉

利用U6启动子转录小发卡RNA介导的RNA干涉是最近发展起来的在哺乳动物细胞中特异性抑制指定基因表达的新技术,已有实验证明它在小鼠畸胎瘤P19等细胞系中具有强烈的抑制基因表达的作用。本文对COS－7细胞系中U6启动子转录GFP基因的小发卡RNA介导的RNA干涉现象进行了研究,结果表明：U6启动子转录的小发卡RNA具有RNA干涉作用,即可以在COS－7细胞中特异性地抑制含有对应序列的基因GFP的表达,这一结果为今后在COS－7细胞系中利用RNA干涉技术研究目的基因的功能奠定了基础。     

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

Background  

DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.     

RNA recognition and cleavage by sequence-specific endoribonucleases

Inactivation of RNA molecules by sequence-specific endoribonucleolytic cleavage is a subtle mechanism by which cells regulate gene expression. Sequence-specific endoribonucleases can recognize and cleave particular phosphodiester bonds confined within hundreds/thousands of chemically similar bonds. Here, we present a comparative analysis of the mechanisms used by endoribonucleases to select and cleave their target RNA molecules. This analysis is based on the very recent molecular details obtained from the structural and/or biochemical studies of nine sequence-specific ribonucleases that target messenger, ribosomal, and transfer RNA molecules. This analysis shows that despite the absence of sequence homologies and the wide diversity of biological sources (prokaryotes, archaea and eukaryotes), the sequence-specific ribonucleases studied here adopt limited structural folds, catalyze their cleavage reactions using a common chemistry and involve a very limited set of amino acids for both RNA binding and processing.     

Accurate cleavage and polyadenylation of exogenous RNA substrate

Purified precursor RNA containing the L3 polyadenylation site of late adenovirus 2 mRNA is accurately cleaved and polyadenylated when incubated with nuclear extract from HeLa cells. The reaction is very efficient; 75% of the precursor is correctly processed. Cleavage is rapidly followed by polymerization of an initial poly(A) tract of approximately 130 nucleotides. Additional adenosine residues are added during further incubation. In the presence of the ATP analog alpha-beta-methylene-adenosine 5' triphosphate, the precursor RNA is cleaved but not polyadenylated, suggesting that processing is not coupled to the synthesis of the initial poly(A) tract. In the absence of free Mg2+, a small RNA of approximately 46 nucleotides is stabilized against degradation. Fingerprint analysis suggests this RNA is produced by endonucleolytic cleavage at the L3 site. Like the in vitro splicing reaction, the in vitro polyadenylation reaction is inhibited by adding antiserum against the small nuclear ribonucleoprotein particle containing U1 RNA.     

Probing ribosome structure by europium-induced RNA cleavage

Divalent metal ions are absolutely required for the structure and catalytic activities of ribosomes. They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metal ion induced RNA cleavages are useful for characterising metal ion binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA of E. coli. The cleavage sites were identified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions. Several Eu3+ cleavage sites, mostly those at which a general metal ion binding site had been already identified, were identical to previously described divalent metal ions. Overall, the Eu3+ cleavages are most similar to the Ca2+ cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+ were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+ cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+ binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.     

Escherichia coli topoisomerase III-catalyzed cleavage of RNA.

Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture. Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium. The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III. The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed. Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.