Substrates For RNase Research Articles

Effects of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine on endonuclease stability and the ability of oligodeoxynucleotide to activate RNase H.

To evaluate an endonuclease resistance property of oligodeoxynucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (Hs) and to elucidate whether a duplex consisting of the ODN analogue and its complementary RNA induces RNase H activity, the ODNs containing the deoxyuridine analogues, Hs, at intervals of one, two, three, four and five natural nucleosides were synthesized. From partial hydrolysis of these ODNs with nuclease S1 (an endonuclease), it was found that the ODNs became more stable towards nucleolytic hydrolysis by the enzyme as the number of H increased. Furthermore, to examine whether the duplexes composed of the ODNs containing Hs and their complementary RNAs are substrates for RNase H or not, the duplexes of these ODNs and their complementary RNA strands were treated with Escherichia coliRNase H. It was found that cleavage of the RNA strands by the enzyme was kinetically affected by the introduction of Hs into the duplexes.

1′,5′‐Anhydrohexitol Oligonucleotides: Hybridisation and Strand Displacement with Oligoribonucleotides, Interaction with RNase H and HIV Reverse Transcriptase

AbstractHexitol nucleic acids (HNAs) with four natural bases form stable and sequence‐selective duplexes with RNA. This was investigated by Tm determinations and gel shift experiments. The CD spectra of an HNA‐RNA duplex show similarities with the CD spectra of the A‐form of dsRNA. Single‐stranded HNAs are able to induce strand displacement in a double‐stranded RNA sequence. An HNA‐RNA duplex is a poor substrate for RNase H, and can inhibit the RNase H‐mediated cleavage of a natural DNA‐RNA substrate. The HNA‐RNA hybrid enhances the activity of HIV reverse transcriptase.

Altered mRNA Splicing and Inhibition of Human E-selectin Expression by an Antisense Oligonucleotide in Human Umbilical Vein Endothelial Cells

We have characterized the mechanism of action of an antisense oligodeoxynucleotide (ASO) targeting human endothelial leukocyte adhesion molecule, E-selectin. ISIS 4730, a 20-base ASO designed to be complementary to a region in the 3'-untranslated region (3'-UTR) of human E-selectin, is a potent and specific inhibitor of both mRNA and protein expression in human umbilical vein endothelial cells. Following treatment with ISIS 4730, a lower molecular weight mRNA (3300 bases) species was detected by Northern blot analysis with a corresponding decrease in the mature E-selectin transcript (3875 bases). The ASO-induced low molecular weight mRNA is stable and remains in the nucleus. We demonstrate that ISIS 4730 targets E-selectin pre-mRNA in the nucleus and promotes cleavage of the pre-mRNA at the hybridization site, resulting in prevention of splicing of the last intron. The change in molecular weight of the E-selectin transcript is the result of loss of the 3'-UTR due to ASO-mediated RNA cleavage and retention of the last intron. Cleavage of the E-selectin pre-mRNA appears to be due to endogenous RNase H or a related enzyme activity.

An RNase P RNA subunit mutation affects ribosomal RNA processing.

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

Antisense Properties of Duplex- and Triplex-Forming PNAs

The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA.

Synthesis and Properties of Diuridine Phosphate Analogues Containing Thio and Amino Modifications.

Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).

Overexpression, Purification, and Properties of Escherichia coli Ribonuclease II

Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded polyribonucleotides processively in the 3' to 5' direction. To understand the role of RNase II in the decay of messenger RNA, a strain overexpressing the rnb gene was constructed. Induction resulted in a 300-fold increase in RNase II activity in crude extracts prepared from the overexpressing strain compared to that of a non-overexpressing strain. The recombinant polypeptide (Rnb) was purified to apparent homogeneity in a rapid, simple procedure using conventional chromatographic techniques and/or fast protein liquid chromatography to a final specific activity of 4,100 units/mg. Additionally, a truncated Rnb polypeptide was purified, solubilized, and successfully renatured from inclusion bodies. The recombinant Rnb polypeptide was active against both [3H]poly(A) as well as a novel (synthetic partial duplex) RNA substrate. The data show that the Rnb polypeptide can disengage from its substrate upon stalling at a region of secondary structure and reassociate with a new free 3'-end. The stalled substrate formed by the dissociation event cannot compete for the Rnb polypeptide, demonstrating that duplexed RNAs lacking 10 protruding unpaired nucleotides are not substrates for RNase II. In addition, RNA that has been previously trimmed back to a region of secondary structure with purified Rnb polypeptide is not a substrate for polynucleotide phosphorylase-like activity in crude extracts. The implications for mRNA degradation and the proposed role for RNase II as a repressor of degradation are discussed.

MicF RNA is a substrate for RNase E

Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichia coli. While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been completely determined. In this study, micF RNA, an antisense RNA found in E. coli and related bacteria, was found to be a substrate for RNase E in vitro. Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures. These results help define a generalized RNase E cleavage/recognition pattern.

MicF RNA is a substrate for RNase E.

Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichia coli. While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been completely determined. In this study, micF RNA, an antisense RNA found in E. coli and related bacteria, was found to be a substrate for RNase E in vitro. Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures. These results help define a generalized RNase E cleavage/recognition pattern.

Evidence for an RNA Binding Region in the Escherichia coli processing Endoribonuclease RNase E

The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.

Precursor of C4 antisense RNA of bacteriophages P1 and P7 is a substrate for RNase P of Escherichia coli.

The C4 repressor of the temperate bacteriophages P1 and P7 inhibits antirepressor (Ant) synthesis and is essential for establishment and maintenance of lysogeny. C4 is an antisense RNA acting on a target, Ant mRNA, which is transcribed from the same promoter. The antisense-target RNA interaction requires processing of C4 RNA from a precursor RNA. Here we show that 5' maturation of C4 RNA in vivo depends on RNase P. In vitro, Escherichia coli RNase P and its catalytic RNA subunit (M1 RNA) can generate the mature 5' end of C4 RNA from P1 by a single endonucleolytic cut, whereas RNase P from the E. coli rnpA49 mutant, carrying a missense mutation in the RNase P protein subunit, is defective in the 5' maturation of C4 RNA. Primer extension analysis of RNA transcribed in vivo from a plasmid carrying the P1 c4 gene revealed that 5'-mature C4 RNA was the predominant species in rnpA+ bacteria, whereas virtually no mature C4 RNA was found in the temperature-sensitive rnpA49 strain at the restrictive temperature. Instead, C4 RNA molecules carrying up to five extra nucleotides beyond the 5' end accumulated. The same phenotype was observed in rnpA+ bacteria which harbored a plasmid carrying a P7 c4 mutant gene with a single C-->G base substitution in the structural homologue to the CCA 3' end of tRNAs. Implications of C4 RNA processing for the lysis/lysogeny decision process of bacteriophages P1 and P7 are discussed.

Synthesis and Self-Cleavage Reaction of a Chimeric Molecule between RNase P-RNA and Its Model Substrate1

We synthesized a chimeric RNA between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a model substrate of the enzyme. The model substrate is the smallest substrate of RNase P, having a simple stem-loop structure. This model substrate was added to the 3'-end of M1 RNA. This chimeric molecule, which we call M1 RNA-MS, is a self-cleaving RNA and is cleaved much more efficiently than the M1 RNA-pre-tRNA, an artificial self-cleaving RNA previously synthesized [Kikuchi et al. (1993) Nucleic Acids Res. 21, 4685-4689], that consists of a full-size tRNA precursor and the M1 RNA. The self-cleavage of M1 RNA-MS at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage). Ca2+ supported the self-cleavage of M1 RNA-MS as effectively as Mg2+, although the self-cleavage of M1 RNA-pre-tRNA proceeded with low efficiency in the presence of Ca2+ as the only metal ion. Future application of the M1 RNA-MS molecule to the in vitro evolution of the M1 RNA and other experiments is proposed.

The use of DNA and RNA oligonucleotides in hybrid structures with longer polynucleotide chains to probe the structural requirements for moloney murine leukemia virus plus strand priming.

Plus strand priming during retroviral reverse transcription requires specific cleavage within the polypurine tract of the viral genome by the reverse transcriptase-associated RNase H. Previously it has been shown that a 190-base RNA-DNA hybrid containing the Moloney murine leukemia virus polypurine tract can serve as a substrate for the priming reaction. To investigate the structural requirements for the reaction, a series of DNA oligonucleotides was hybridized to the 190-base single-stranded RNA and tested as substrates for RNase H. At low enzyme concentrations, the sites of cleavage are located 17-23 nucleotides from the 3'-end of the DNA oligonucleotide, consistent with the observations of others that binding of the DNA polymerase at a primer terminus fixes the position of cleavage by RNase H. At higher enzyme concentrations, additional cleavages are observed in the RNA 3' of these sites, but there is no preference for cleavage at the plus strand origin. In contrast to the results with DNA oligonucleotides, hybridization of RNA oligonucleotides containing the polypurine tract to the 190-base single-stranded DNA generates substrates that are cleaved at the origin and efficiently extended into DNA. An RNA oligonucleotide hybridized downstream of the polypurine tract is cleaved but not extended. These results support the view that RNase H cleavage to generate the plus strand primer is uncoupled from minus strand DNA synthesis.

αβ Chimeric Antisense Oligonucleotides: Synthesis and Nuclease Resistance in Biological Media

A new type of chimeric oligonucleotides composed of alpha- and beta-sections is described. The sequence of eight beta-nucleotides flanked at 3'- or/and 5'-ends by nuclease-resistant alpha-oligonucleotides has been chosen as an effector domain to form a substrate for RNase H. The synthesized oligonucleotides are complementary to the translation initiation site of the pim protooncogene mRNA. We used the chemical ligation method to prepare the chimeric oligonucleotides. The thermal stability of heteroduplexes formed by the alpha beta oligonucleotides with a complementary strand is not significantly altered compared to that of their beta-analogs. These oligonucleotides promote efficient RNase H-mediated cleavage of pim mRNA. Among the alpha beta oligonucleotides studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octanucleotide proved to be the most resistant to nucleolytic digestion in human plasma, calf serum, and murine fibroblast lysate. This alpha beta oligonucleotide directs more specific RNA cleavage by RNase H than its beta beta counterpart.

Role of the D arm and the anticodon arm in tRNA recognition by eubacterial and eukaryotic RNase P enzymes.

Truncated precursor tRNAs lacking the D arm or anticodon arm were studied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm still allowed 5'-processing by E. coli RNase P, but strongly impaired maturation by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activities from all three organisms. Inhibition kinetics and gel retardation studies showed that deletion of the D arm leads to low-affinity binding to E. coli RNase P RNA (M1 RNA). However, the E. coli enzyme appears to form sufficiently strong contacts in the region of the T arm, acceptor stem, and CCA terminus to still allow productive enzyme-substrate interaction even in the absence of the structural contribution provided by the D arm. Pb(2+)-induced hydrolysis of a tRNAGly from T. thermophilus gave identical cleavage patterns in the D arm and anticodon loop in the absence and presence of E. coli M1 RNA, whereas lead hydrolysis was strongly reduced at the CUCCAA 3'-terminus due to the presence of the enzyme.

Antisense oligoribonucleotides and RNase P. A great potential

This paper presents the possible — but at present mostly hypothetical — applications of RNase P for the specific inactivation of target RNAs. The natural substrates for RNase P are pre-tRNAs. The enzyme can also recognize and cleave smaller model substrates. These are simple hairpins for bacterial RNase P whereas the requirements for eukaryotic RNase P are more complex and less well understood. It is possible to split the RNase P substrates into two separate RNA molecules. One part of the split substrate RNA contains the RNase P cleavage site and the 5′-terminal half of the acceptor stem, embedded in a large target RNA. The other part of the substrate RNA provides the 3′-terminal half of the acceptor stem and it serves as antisense sequence (‘external guide sequence’). Both parts can hybridize and reconstitute a functional substrate for RNase P; this results in cleavage of the target RNA by RNase P. The properties of this system are presented and advantages and problems discussed.

Important 2′-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli

The role of 2′-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2′-hydroxyl groups of nucleotides at positions −1 and −2 in the leader sequence and at position 1, as well as at the first C in the 3′-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2′-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg 2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg 2+-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus.

We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.

Kinetics of the processing of the precursor to 4·5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4·5 S RNA (p4·5S). The overall efficiency of cleavage of p4·5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4·5S as substrate has a much lower K m value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4·5S. A model hairpin substrate, based on the sequence of p4·5S, is cleaved with greater efficiency that the parent molecule. The 3′-terminal CCC sequence of p4·5 S may be as important for cleavage of this substrate as the 3′-terminal CCA sequence is for cleavage of tRNA precursors.