Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Substrate structure requirements of the Pac1 ribonuclease from Schizosaccharmyces pombe.

The Pac1 ribonuclease of Schizosaccharomyces pombe is a member of the RNase III family of double-strand-specific ribonucleases. To examine RNA structural features required for efficient cleavage by the Pac1 RNase, we tested a variety of double-stranded and hairpin RNAs as substrates for the enzyme. The Pac1 RNase required substrates that have a minimal helix length of about 20 base pairs. The enzyme cut both strands of the helix at sites separated by two base pairs. However, Pac1 was also able to make a single-stranded cleavage within an internal bulge of an authentic Escherichia coli substrate at the same site chosen by RNase III. Pac1 efficiently degraded the structurally complex adenovirus VA RNA(I), but was inactive against the short HIV-1 TAR RNA hairpin. These results indicate that the Pac1 RNase prefers straight, perfect helices, but it can tolerate internal bulges that do not distort the helix severely. Like its homologue from Saccharomyces cerevisiae, the Pac1 RNase cleaved at two in vivo RNA processing sites in a hairpin structure in the 3' external transcribed spacer of the S. pombe pre-rRNA, suggesting a role for the enzyme in rRNA maturation.     

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.     

S. pombe pac1+, whose overexpression inhibits sexual development, encodes a ribonuclease III-like RNase.

The Schizosaccharomyces pombe pac1 gene is a multicopy suppressor of the pat1 temperature-sensitive mutation, which directs uncontrolled meiosis at the restrictive temperature. Overexpression of the pac1 gene had no apparent effect on vegetative growth but inhibited mating and sporulation in wild type S. pombe cells. In such cells, expression of certain genes required for mating or meiosis was inhibited. The pac1 gene is essential for vegetative cell growth. The deduced pac1 gene product has 363 amino acids. Its C-terminal 230 residues revealed 25% amino acid identity with ribonuclease III, an enzyme that digests double-stranded RNA and is involved in processing ribosomal RNA precursors and certain mRNAs in Escherichia coli. The pac1 gene product could degrade double-stranded RNA in vitro. These observations establish the presence of a RNase III homolog in eukaryotic cells. The pac1 gene product probably inhibits mating and meiosis by degrading a specific mRNA(s) required for sexual development. It is likely that mRNA processing is involved in the regulation of sexual development in fission yeast.     

Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage

Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.     

RAC protein directs the complete removal of the 3' external transcribed spacer by the Pac1 nuclease

In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS. Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence. The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase-binding domains

Previous work has detected an RNase E-like endoribonucleolytic activity in cell extracts obtained from Streptomyces. Here, we identify a Streptomyces coelicolor gene, rns, encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E-like regulation of plasmid copy number in E. coli. However, notwithstanding its complementation of rne-deleted E. coli, RNase ES did not accurately process 9S rRNA from E. coli. Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor. Deletion analysis mapped the catalytic domain of RNase ES near its centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) - a major component of E. coli RNase E-based degradosomes. The interacting arginine- and proline-rich segments resemble the C-terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E-like enzymatic activity, and suggest the existence of degradosome-like complexes in Gram-positive bacteria.     

Functional Conservation of RNase III-like Enzymes: Studies on a Vibrio vulnificus Ortholog of Escherichia coli RNase III

Bacterial ribonuclease III (RNase III) belongs to the RNase III enzyme family, which plays a pivotal role in controlling mRNA stability and RNA processing in both prokaryotes and eukaryotes. In the Vibrio vulnificus genome, one open reading frame encodes a protein homologous to E. coli RNase III, designated Vv-RNase III, which has 77.9 % amino acid identity to E. coli RNase III. Here, we report that Vv-RNase III has the same cleavage specificity as E. coli RNase III in vivo and in vitro. Expressing Vv-RNase III in E. coli cells deleted for the RNase III gene (rnc) restored normal rRNA processing and, consequently, growth rates of these cells comparable to wild-type cells. In vitro cleavage assays further showed that Vv-RNase III has the same cleavage activity and specificity as E. coli RNase III on RNase III-targeted sequences of corA and mltD mRNA. Our findings suggest that RNase III-like proteins have conserved cleavage specificity across bacterial species.     

Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates

The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence. Instead, the [4/5] asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop. The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex. The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry. The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.     

RNase III controls the degradation of corA mRNA in Escherichia coli

In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.     

Bacteriophage P1 genes involved in the recognition and cleavage of the phage packaging site (pac).

The packaging of bacteriophage P1 DNA is initiated by cleavage of the viral DNA at a specific site, designated pac. The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report. By sequencing wild-type P1 DNA and DNA derived from various P1 amber mutants that are deficient in pac cleavage, two distinct genes, referred to as pacA and pacB, were identified. These genes appear to be coordinately transcribed with an upstream P1 gene that encodes a regulator of late P1 gene expression (gene 10). pacA is located upstream from pacB and contains the 161 base-pair pac cleavage site. The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa, respectively. These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter. Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the P1 packaging site, whereas those lacking either protein are not. However, the two defective extracts can complement each other to restore functional pac cleavage activity. Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the P1 packaging site. PacB extracts also contain a second P1 protein that is encoded within the pacB gene. We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB. Its role, if any, in pac cleavage is yet to be determined.     

Studies on a Vibrio vulnificus Functional Ortholog of Escherichia coli RNase E Imply a Conserved Function of RNase E-like Enzymes in Bacteria

RNase E (Rne) plays a key role in the processing and degradation of RNA in Escherichia coli. In the genome of Vibrio vulnificus, one open reading frame potentially encodes a protein homologous to E. coli RNase E, designated RNase EV, which N-terminal (1-500 amino acids) has 86.4% amino acid identity to the N-terminal catalytic part of RNase E (N-Rne). Here, we report that both the full-length and the N-terminal part of RNase EV (N-RneV) functionally complement E. coli RNase E and their expression consequently supports normal growth of RNase E-depleted E. coli cells. E. coli cells expressing N-RneV showed copy numbers of ColE1-type plasmid similar to that of E. coli cells expressing N-Rne, indicating in vivo ribonucleolytic activity of N-RneV on RNA I, an antisense regulator of ColE1-type plasmid replication. In vitro cleavage assays further showed that N-RneV has cleavage activity and specificity of RNase E on RNase E-targeted sequence of RNA I (BR13). Our findings suggest that RNase E-like proteins have conserved enzymatic properties that determine substrate specificity across species.     

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.     

Effects of C5 protein on Escherichia coli RNase P catalysis with a precursor tRNA(Phe) bearing a single mismatch in the acceptor stem

Escherichia coli RNase P, an RNA-processing enzyme that cleaves precursor tRNAs to generate the mature 5'-end, is composed of a catalytic component (M1 RNA) and a protein cofactor (C5 protein). In this study, effects of C5 protein on the RNase P catalysis with a precursor E. coli tRNA(Phe) having a single mismatch in the acceptor stem were examined. This mutant precursor unexpectedly generated upstream cleavage products at the -8 position as well as normal cleavage products at the +1 position. The cleavage at the -8 position was essentially effective only in the presence of C5 protein. Possible secondary structures for cleavage at the -8 position deviate significantly from the structures of the known RNase P substrates, implying that C5 protein can allow the enzyme to broaden the substrate specificity more than previously appreciated.