Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes

Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli. We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro. However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated. Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II. Moreover, both ATP and a non-hydrolyzable analog, ATPgammaS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required. Alternatively, the rpsT mRNA can be degraded in vitro by a second 3'-decay pathway which is dependent on PAP I, PNPase and ATP alone. Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3' termini. Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined.     

Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5' to cleavage sites

The endoribonuclease RNase E is believed to initiate the degradation of many mRNAs in Escherichia coli, yet the mechanism by which it recognizes cleavage sites is poorly understood. We have prepared derivatives of the mRNA encoding ribosomal protein S20 which contain a single major RNase E cleavage site at residues 300/301 preceded by variable 5' extensions. Three of these RNAs are cleaved in vitro with significantly reduced efficiencies relative to the intact S20 mRNA by both crude RNase E and pure Rne protein (endonuclease component of RNase E). In all three substrates as well as in the full-length mRNA the major cleavage site itself remains single-stranded. One such substrate (t84D) contains a 5' stem-loop structure characterized by three noncanonical A-G pairs. Removal or denaturation of the stem restores efficient cleavage at the major RNase E site. The other two contain single-stranded 5'-termini but apparently lack cleavage sites near the termini. Our data show that sensitivity to RNase E can be influenced by distant structural motifs in the RNA and also suggest a model in which the initial recognition and cleavage of a substrate near its 5' end facilitates sequential cleavages at more distal sites. The model implies that RNase E contains at least a dimer of the Rne subunit and that the products of the first cleavage are retained by Rne prior to the second cleavage.     

Cleavage of single strand RNA adjacent to RNA-DNA duplex regions by Escherichia coli RNase H1

RNase H1 from Escherichia coli cleaves single strand RNA extending 3' from an RNA-DNA duplex. Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9-17 nucleotides were designed to create overhanging single strand RNA regions extending 5' and 3' from the RNA-DNA duplex. Digestion of single strand RNA was observed exclusively within the 3' overhang region and not the 5' overhang region. RNase H digestion of the 3' overhang region resulted in digestion products with 5'-phosphate and 3'-hydroxyl termini. The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex. RNase H digestion of the 3' overhang region was not observed for a substrate that contained a 2'-methoxy antisense strand. The introduction of 3 deoxynucleotides at the 5' terminus of the 2'-methoxy antisense oligonucleotide resulted in cleavage. These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.     

Potent and selective inhibition of gene expression by an antisense heptanucleotide

Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Identification and partial purification of human double strand RNase activity. A novel terminating mechanism for oligoribonucleotide antisense drugs

We have identified a double strand RNase (dsRNase) activity that can serve as a novel mechanism for chimeric antisense oligonucleotides comprised of 2'-methoxy 5' and 3' "wings" on either side of an oligoribonucleotide gap. Antisense molecules targeted to the point mutation in codon 12 of Harvey Ras (Ha-Ras) mRNA resulted in a dose-dependent reduction in Ha-Ras RNA. Reduction in Ha-Ras RNA was dependent on the oligoribonucleotide gap size with the minimum gap size being four nucleotides. An antisense oligonucleotide of the same composition, but containing four mismatches, was inactive. When chimeric antisense oligonucleotides were prehybridized with 17-mer oligoribonucleotides, extracts prepared from T24 cells, cytosol, and nuclei resulted in cleavage in the oligoribonucleotide gap. Both strands were cleaved. Neither mammalian nor Escherichia coli RNase HI cleaved the duplex, nor did single strand nucleases. The dsRNase activity resulted in cleavage products with 5'-phosphate and 3'-hydroxyl termini. Partial purification of dsRNase from rat liver cytosolic and nuclear fractions was effected. The cytosolic enzyme was purified approximately 165-fold. It has an approximate molecular weight of 50,000-65,000, a pH optimum of approximately 7.0, requires divalent cations, and is inactivated by approximately 300 mM NaCl. It is inactivated by heat, proteinase K, and also by a number of detergents and several organic solvents.     

The 2-5A system in viral infection and apoptosis

The 2-5A system is an established endogenous antiviral pathway. Interferon treatment of cells leads to an increase in basal, but latent, levels of 2-5A-dependent RNase (RNase L) and the family of 2'-5' oligoadenylate synthetases (OAS). Double-stranded RNA, thought to be derived from viral replication intermediates, activates OAS. Activated OAS converts ATP into unusual short 2'-5' linked oligoadenylates called 2-5A [ppp5'(A2'p5')2A]. The 2-5A binds to and activates RNase L which cleaves single stranded RNA with moderate specificity for sites 3' of UpUp and UpAp sequences, and thus leads to degradation of cellular rRNA. During apoptosis, generalized cellular RNA degradation, distinct from the differential expression of mRNA species that may regulate specific gene expression during apoptosis, has been observed. The mechanism of RNA breakdown during apoptosis has been commonly considered a non-specific event that reflects the generalized shut down of translation and homeostatic regulation during cell death. Due to the similar RNA degradation that occurs during both apoptosis and viral infection we investigated the potential role of RNase L in apoptosis. To investigate whether RNase L activity could lead to apoptosis, NIH3T3 cells were transfected with a lac-inducible vector containing the human RNase L gene. Treatment of these cells with isopropylthiogalactoside (IPTG) caused loss of cell viability that was confirmed as an apoptotic cell death by morphological and biochemical criteria. Similarly, specific allosteric activation of endogenous RNase L by introduction of 2-5A directly into L929 cells also induced apoptosis. In L929 cells poly(I).poly(C) treatment in combination with interferon caused an increase in apoptosis whereas neither interferon or double stranded RNA alone altered cell viability. Therefore, increased expression or activation of RNase L causes apoptosis. Inhibition of RNase L, specifically with a dominant negative mutant, suppressed poly(I)Ypoly(C)-induced apoptosis in interferon-primed fibroblasts. Poliovirus, a picornovirus with a single-stranded RNA genome, causes apoptosis of HeLa cells. Expression of the dominant negative inhibitor of RNase L in HeLa prevented virus-induced apoptosis and maintained cell viability. Thus, reduction or inhibition of RNase L activity prevents apoptosis. Both apoptosis and the 2-5A system can provide defense against viral infection in multicellular organisms by preventing production and therefore spread of progeny virus. RNase L appears to function in both mechanisms, therefore, initiation of apoptosis may be one mechanism for the antiviral activity of the 2-5A system.     

Multiple methylated cap sequences in adenovirus type 2 early mRNA

The methylated constituents of early adenovirus 2 mRNA were studied. RNA was isolated from polyribosomes of cells double labeled with [methyl-3H]methionine and 32PO4 from 2 to 7 g postinfection in the presence of cycloheximide. Cycloheximide ensures that methylation and processing are performed by preexisting host cell enzymes. RNA was fractionated into polyadenylic [poly(A)]+ and poly(A)- molecules using poly(U)-Sepharose, and undergraded virus-specific RNA was isolated by hybridization to viral DNA in 50% formamide at 37 degrees C. Viral mRNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea. Two 3H-labeled RNase T2-resistant oligonucleotide fractions with charges between -5 and -6 were obtained, consistent with two classes of 5' terminal methyl "cap" structures, m7G(5')ppp(5')NmpNp (cap 1) and m7G(5')ppp(5')NmNmpNp (cap 2) (Nm is a ribose 2'-O-methylation). The putative cap 1 contains all the methylated constituents of cap 1 plus Cm. The molar ratios of m7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap 2, consistent with the proposed cap structures. Most significant, compositional analysis indicates four different cap 1 structures and at least three different cap 2 structures. Thus there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNA can have more than one 5' terminus. In addition to methylated caps, early mRNA contains internal base methylations, exclusively as m6A, as shown by analyses of the mononucleotide (-2 charge) fraction. m6A was present in the ratio of 1 mol of m6Ap per 450 nucleotides. Thus viral mRNA molecules contain two to three internal m6A residues per methyl cap, since there is on the average 1 cap per 1,250 nucleotides.     

Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 5'-rUUGGCG-3' motif in the loop sequence

The sequence 5'-rUUGGCG-3' is conserved within the loop regions of antisense RNAs or their targets involved in replication of various prokaryotic plasmids. In IncIalpha plasmid ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop called structure I within RepZ mRNA contains this hexanucleotide sequence, and comprises the target site for the antisense Inc RNA. In this report, we find that the base pairing interaction at the 5'-rGGC-3' sequence in the hexanucleotide motif is important for interaction between Inc RNA and structure I. In addition, the 21-base loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top. The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner. On the other hand, the upper domain of the Inc RNA stem-loop is loosely structured, and maintaining the loop sequence single-stranded is important for the intermolecular interaction. Based on these results, we propose that a structural feature in the loop I domain, conferred probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a complementary, single-stranded RNA. This model also explains how the RepZ mRNA pseudoknot, described in the accompanying paper (Asano, K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825) is formed specifically with structure I. A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is discussed.     

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.     

Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase

2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.