Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

A hybrid ribonuclease H. A novel RNA cleaving enzyme with sequence-specific recognition.

A hybrid enzyme which site-specifically hydrolyzes RNA was created by covalently linking an oligodeoxyribonucleotide to Escherichia coli ribonuclease HI, an enzyme which specifically cleaves RNA moiety of DNA/RNA hybrids. A cysteine residue was substituted for Glu135 by site-directed mutagenesis in the mutant enzyme, in which all 3 free cysteine residues were replaced by alanine (Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochem. J. 271, 59-66), and coupled with a maleimide group, which is attached to the 5' terminus of the nonadeoxyribonucleotide (5'-GTCATCTCC-3') with a flexible tether. The resulting hybrid enzyme, d9-C135/RNase H, cleaved the phosphodiester bond between the fifth and sixth residues of the complementary nonaribonucleotide, without addition of the oligodeoxyribonucleotide. The nonaribonucleotide is cleaved by the wild-type or unmodified mutant enzyme only when the complementary oligodeoxyribonucleotide is present. When the kinetic parameters of the hybrid enzyme for the hydrolysis of the nonaribonucleotide were compared with those of the unmodified mutant enzyme for the hydrolysis of the nonanucleotide duplex, the hybrid enzyme exhibited a 7- and 4-fold decreases in the Km and kcat values, respectively, indicating that it performs multiple turnovers and has a sufficiently high hydrolytic activity. Hybrid ribonucleases H with various oligodeoxyribonucleotides in size and sequence, therefore, might be used as excellent tools for structural and functional studies of RNA.     

Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H

To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Specific inhibition of capped mRNA translation in vitro by m7G5''pppp5''G and m7G5''pppp5''m7G.

A unique set of diguanosine cap analogues containing a 5'-5' tetraphosphate linkage instead of the normal triphosphate was synthesized by chemical methylation of G5'pppp5'G. Both 7-methylguanosine products, m7G5'pppp5'G and m7G5'pppp5'm7G, acted as potent inhibitors of capped brome mosaic virus (BMV) RNA translation in the homologous wheat germ protein synthesis system. Inhibition of in vitro protein synthesis required the presence of the 7-methyl group on guanosine and was specific for capped mRNA. In comparison with the partial cap analogue, m7GTP, the methylated diguanosine tetraphosphate structures were 25-50 fold more potent inhibitors of in vitro protein synthesis. Analysis of the in vitro translation products of the four species of BMV RNA showed a differential sensitivity to inhibition by m7G5'pppp5'm7G.     

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

Probing DNA single strands for single-base bulges with neocarzinostatin chromophore.

Neocarzinostatin chromophore (NCS-Chrom) induces strong cleavage at a single site (C3) in the single-stranded and 5' (32)P-end-labeled 13-mer GCCAGATTTGAGC in a reaction dependent on a thiol. By contrast, in the duplex form of the same 13-mer, strand cleavage occurs only at the T and A residues, and C3 is not cleaved. To determine the minimal structural requirement(s) for C3 cleavage in the single-stranded oligomer, several deletions and mutations were made in the 13-mer. A 10-mer (GCCAGAGAGC) derived from the 13-mer by deletion of the three T residues was also cleaved exclusively at C3 by NCS-Chrom, generating fragments having 5' phosphate ends. That the cleavage at C3 is initiated by abstraction of its 5' hydrogen is confirmed in experiments using 3' (32)P-end-labeled 10-mer. The competent 13-mer and 10-mer were assigned hairpin structures with a stem loop and a single bulged out A base, placing C3 across from and 3' to the bulge. Removal of the bulged A base from the 13-mer and the 10-mer resulted in complete loss of cutting activity, proving that it is the essential determinant in competent substrates. Studies of thiol post-activated NCS-Chrom binding to the DNA oligomers show that the drug binds to the bulge-containing 13-mer (K(d) = 0.78 microM) and the 10-mer (K(d) = 1.11 microM), much more strongly than to the 12-mer (K(d) = 20 microM) and the 9-mer (K(d) = 41 microM), lacking the single-base bulge. A mutually induced-fit between NCS-Chrom and the oligomer resulting in optimal stabilization of the drug-DNA complex is proposed to account for the site-specific cleavage at C3. These studies establish the usefulness of NCS-Chrom as a probe for single-base bulges in DNA.     

Preliminary physical mapping of RNA-RNA linkages in the genomic RNA of Moloney murine leukemia virus

Retrovirus particles contain two copies of their genomic RNA, held together in a dimer by linkages which presumably consist of a limited number of base pairs. In an effort to localize these linkages, we digested deproteinized RNA from Moloney murine leukemia virus (MLV) particles with RNase H in the presence of oligodeoxynucleotides complementary to specific sites in viral RNA. The cleaved RNAs were then characterized by nondenaturing gel electrophoresis. We found that fragments composed of nucleotides 1 to 754 were dimeric, with a linkage as thermostable as that between dimers of intact genomic RNA. In contrast, there was no stable linkage between fragments consisting of nucleotides 755 to 8332. Thus, the most stable linkage between monomers is on the 5' side of nucleotide 754. This conclusion is in agreement with earlier electron microscopic analyses of partially denatured viral RNAs and with our study (C. S. Hibbert, J. Mirro, and A. Rein, J. Virol. 78:10927-10938, 2004) of encapsidated nonviral mRNAs containing inserts of viral sequence. We obtained similar results with RNAs from immature MLV particles, in which the dimeric linkage is different from that in mature particles and has not previously been localized. The 5' and 3' fragments of cleaved RNA are all held together by thermolabile linkages, indicating the presence of tethering interactions between bases 5' and bases 3' of the cleavage site. When RNAs from mature particles were cleaved at nucleotide 1201, we detected tethering interactions spanning the cleavage site which are intramonomeric and are as strong as the most stable linkage between the monomers.     

Involvement of precursor-specific segments in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

In vitro maturation of precursor 5S ribosomal RNA (p5A) from Bacillus subtilis effected by RNase M5 yields mature 5S RNA (m5, 116 nucleotides), and 3' precursor-specific segment (42 nucleotides), and a 5' precursor-specific segment (21 nucleotides) (Sogin, M.L., Pace, B., and Pace, N.R. (1977), J. Biol. Chem. 252, 1350). Limited digestion of p5A with RNase T2 introduces a single scission at position 60 of the molecule; m5 is cleaved at the corresponding nucleotide residue. The complementary "halves" of the molecules could be isolated from denaturing polyacrylamide gels. The isolated fragments of p5A are not substrates for RNase M5, suggesting that some recognition elements can be utilized by RNase M5 only when presented in double-helical form. In exploring the involvement of the precursor-specific segments in the RNase M5-p5A interaction, substrate molecules lacking the 3' or 5' precursor-specific segment were constructed by reannealing complementary "halves" from p5A and m5 RNA. The artificial substrate lacking the 5'-terminal precursor segment was cleaved very much more slowly than the lacking t' segment; the 5' precursor-specific segment therefore contains one or more components recognized by RNase M5 during its interaction with the p5A substrate.     

The use of thioglycolate to distinguish between 3'' AP (apurinic/apyrimidinic) endonucleases and AP lyases.

Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase). For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex: deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate:unsaturated sugar-5-phosphate adduct (indicating a cleavage by beta-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease. We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by beta-elimination is an irreversible reaction. We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the beta-elimination reaction from undergoing other modifications that complicate the interpretation of the results.     

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.     

Specific cleavage of target RNAs from HIV-1 with 5' half tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can be converted to an RNA cutter that recognizes four bases, with about a 65-nt 3'-truncated tRNA(Arg) or tRNA(Ala). The 3'-truncated tRNA recognizes the target RNA via four base pairings between the 5'terminal sequence and a sequence 1-nt upstream of the cleavage site, resulting in a pre-tRNA-like complex (Nashimoto M, 1995, Nucleic Acids Res 23:3642-3647). Here I developed a general method for more specific RNA cleavage using 3' tRNase. In the presence of a 36-nt 5' half tRNA(Arg) truncated after the anticodon, 3' tRNase cleaved the remaining 56-nt 3' half tRNA(Arg) with a 19-nt 3' trailer after the discriminator. This enzyme also cleaved its derivatives with a 5' extra sequence or nucleotide changes or deletions in the T stem-loop and extra loop regions, although the cleavage efficiency decreases as the degree of structural change increases. This suggests that any target RNA can be cleaved site-specifically by 3'tRNase in the presence of a 5' half tRNA modified to form a pre-tRNA-like complex with the target. Using this method, two partial HIV-1 RNA targets were cleaved site-specifically in vitro. These results also indicate that the sequence and structure of the T stem-loop domain are important, but not essential, for the recognition of pre-tRNAs by 3' tRNase.     

Conversion of mammalian tRNA 3' processing endoribonuclease to four-base-recognizing RNA cutters.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) activities in mammalian cell extracts require both protein and 3' truncated tRNA, species of which direct their substrate sequence specificity. Computer analysis for searching possible base pairing between substrate RNAs and their corresponding 3' truncated tRNA, suggested a unified model for substrate recognition mechanism, in which a four-nucleotide (nt) sequence in the target tRNAs 1 nt upstream of their cleavage site, base pairs with the 5' terminal 4 nt sequence of their corresponding 3' truncated tRNA. This model was supported by experiments with several RNA substrates containing a substituted nucleotide in the target 4 nt sequence. In this model, the tRNA substrates and their corresponding 3' truncated tRNA form a complex resembling a 5' processed tRNA precursor containing a 3' trailer, suggesting that the protein component of RNase 65 is identical to tRNA 3' processing endoribonuclease (3' tRNase). Actually, 3' tRNase purified from pig liver cleaved the target RNAs at the expected sites only in the presence of their corresponding 3' truncated tRNA. These results show that the 3' tRNase can be converted to 4 nt specific RNA cutters using the 3' truncated tRNAs.     

Anomalous RNA substrates for mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA. To investigate the interaction between 3' tRNase and substrates, we tested various anomalous pre-tRNA-like complexes for cleavage by pig 3' tRNase. We examined how base mismatches in the acceptor stem affect 3' tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3' tRNase was able to recognize complexes between target RNAs and 5'-half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3' tRNase in the presence of appropriate 5'-half tRNAs. We also demonstrated that an RNA complex of lin-4 and lin-14 from Caenorhabditis elegans can be recognized and cleaved by pig 3' tRNase.     

RNA heptamers that direct RNA cleavage by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.     

Stimulation of RecA-mediated cleavage of phage phi 80 cI repressor by deoxydinucleotides

The presence of either deoxyguanylyl-(3'----5')-deoxyguanosine (d(G-G] or deoxyadenylyl-(3'----5')-deoxyguanosine (d(A-G] greatly stimulates cleavage of the phage phi 80 cI repressor mediated by the Escherichia coli RecA protein in vitro. No other deoxydinucleoside monophosphate or riboguanylyl-(3'----5')-guanosine (r(G-G] affects the cleavage reaction. Neither the cleavage site of the phi 80 cI repressor nor the requirement for single-stranded DNA and ATP for cleavage is altered by d(G-G). Photoaffinity labeling experiments with 32P-labeled 5'-phosphoryl deoxyguanylyl deoxyguanosine (pd(G-G], which also stimulates cleavage, show that pd(G-G) bound to the repressor under the conditions in which the repressor is cleaved by RecA protein. The binding increases the affinity of the repressor for RecA protein and thus greatly stimulates repressor cleavage. The cleavage reactions of LexA and lambda cI repressors by RecA protein are not affected by d(G-G).     

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.     

Cleavage reaction of a synthetic oligoribonucleotide corresponding to the autocleavage site of a precursor RNA from bacteriophage T4

A fragment (GUUUCGUACAAAC) having a consensus sequence for the self-cleavage domain in a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Sp1; precursor of species 1) was chemically synthesized and found to be cleaved either between CA (139-140) or between UA (137-138) in the presence of monovalent cations and a non-ionic detergent. The cleaved products had 5'-hydroxyl and 3'-phosphate groups, of which some were in the form 2',3'-cyclic phosphates.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.