Bacillus subtilis YhaM,a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria

A strain of Bacillus subtilis lacking two 3'-to-5' exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3'-to-5' exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn(2+) (or Co(2+)), was inactive in the presence of Mg(2+), and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn(2+)-dependent exoribonuclease. YhaM protein has a C-terminal HD domain, found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal OB-fold, present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.     

5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA

Although the primary mechanism of eukaryotic messenger RNA decay is exoribonucleolytic degradation in the 5'-to-3' orientation, it has been widely accepted that Bacteria can only degrade RNAs with the opposite polarity, i.e. 3' to 5'. Here we show that maturation of the 5' side of Bacillus subtilis 16S ribosomal RNA occurs via a 5'-to-3' exonucleolytic pathway, catalyzed by the widely distributed essential ribonuclease RNase J1. The presence of a 5'-to-3' exoribonuclease activity in B. subtilis suggested an explanation for the phenomenon whereby mRNAs in this organism are stabilized for great distances downstream of "roadblocks" such as stalled ribosomes or stable secondary structures, whereas upstream sequences are never detected. We show that a 30S ribosomal subunit bound to a Shine Dalgarno-like element (Stab-SD) in the cryIIIA mRNA blocks exonucleolytic progression of RNase J1, accounting for the stabilizing effect of this element in vivo.     

The yvaJ gene of Bacillus subtilis encodes a 3'-to-5' exoribonuclease and is not essential in a strain lacking polynucleotide phosphorylase

Studies of Bacillus subtilis RNases that are involved in mRNA degradation reveal a different pattern from that of Escherichia coli. A strain lacking polynucleotide phosphorylase, the major 3'-to-5' exoribonuclease activity in cell extracts, is viable. Here, we show that the B. subtilis yvaJ gene encodes a second 3'-to-5' exoribonuclease. A strain lacking both of these RNases grows slowly but is viable. The existence of another, as yet unknown, 3'-to-5' exoribonuclease in B. subtilis is suggested.     

Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping

The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.     

A polypurine sequence that acts as a 5' mRNA stabilizer in Bacillus subtilis.

A segment of early RNA from Bacillus subtilis bacteriophage SP82 was shown to function as a 5' stabilizer in B. subtilis. Several heterologous RNA sequences were stabilized by the presence of the SP82 sequence at the 5' end, and expression of downstream coding sequences was increased severalfold. The SP82 RNA segment encodes a B. subtilis RNase III cleavage site, but cleavage by B. subtilis RNase III was not required for stabilization. The sequence that specifies 5' stabilizer function was localized to a polypurine sequence that resembles a ribosome binding site. The ability of the SP82 sequence to stabilize downstream RNA was dependent on its position relative to the 5' end of the RNA. These results demonstrate the existence of a new type of 5' stabilizer in B. subtilis and indicate that attack at the 5' end is a principal mechanism for initiation of mRNA decay in B. subtilis.     

Doing it in reverse: 3'-to-5' polymerization by the Thg1 superfamily

The tRNA(His) guanylyltransferase (Thg1) family of enzymes comprises members from all three domains of life (Eucarya, Bacteria, Archaea). Although the initial activity associated with Thg1 enzymes was a single 3'-to-5' nucleotide addition reaction that specifies tRNA(His) identity in eukaryotes, the discovery of a generalized base pair-dependent 3'-to-5' polymerase reaction greatly expanded the scope of Thg1 family-catalyzed reactions to include tRNA repair and editing activities in bacteria, archaea, and organelles. While the identification of the 3'-to-5' polymerase activity associated with Thg1 enzymes is relatively recent, the roots of this discovery and its likely physiological relevance were described ≈ 30 yr ago. Here we review recent advances toward understanding diverse Thg1 family enzyme functions and mechanisms. We also discuss possible evolutionary origins of Thg1 family-catalyzed 3'-to-5' addition activities and their implications for the currently observed phylogenetic distribution of Thg1-related enzymes in biology.     

Selective protection of 5' ... GGCC ... 3' and 5' ... GCNGC ... 3' sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA.

The DNA of Bacillus subtilis bacteriophage SP10 is partially resistant to cleavage and methylation in vitro by restriction enzyme R . BsuRI and its cognate methylase even though greater than 20 copies of the target sequence, 5' ... GGCC ... 3', are present on the phage genome. YThy, a hypermodified oxopyrimidine that replaces a fraction of the thymine residues in SP10 DNA, was responsible for this protection, since YThy-free DNA was no longer resistant. Sites that were normally resistant could nevertheless be cleaved or methylated in vitro if the salt concentration was reduced or dimethyl sulfoxide was added to the reaction buffer. Analysis of the termini produced by cleavage suggested that resistant sites occurred in the sequence 5' ... GGCC-YThy ... 3', whereas sensitive sites, of which there were only two per genome, occurred in the sequence 5' ... GGCCG ... 3'. These in vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. They also suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation. YThy also strongly protected 5' ... GCNGC ... 3' (R . Fnu4HI) sequences on SP10 DNA, but the biological relevance of this protection is unclear.     

Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.     

Identification of an RNase J ortholog in Sulfolobus solfataricus: implications for 5'-to-3' directional decay and 5'-end protection of mRNA in Crenarchaeota

In both Bacteria and Eukaryotes, degradation is known to start at the 5' and at the 3' extremities of mRNAs. Until the recent discovery of 5'-to-3' exoribonucleases in hyperthermophilic Euryarchaeota, the exosome was assumed to be the key enzyme in mRNA degradation in Archaea. By means of zymogram assays and bioinformatics, we have identified a 5'-to-3' exoribonuclease activity in the crenarchaeum Sulfolobus solfataricus (Sso), which is affected by the phosphorylation state of the 5'-end of the mRNA. The protein comprises typical signature motifs of the β-CASP family of metallo-β-lactamases and was termed Sso-RNAse J. Thus, our study provides the first evidence for a 5'-to-3' directional mRNA decay pathway in the crenarchaeal clade of Archaea. In Bacteria the 5'-end of mRNAs is often protected by a tri-phosphorylated 5'-terminus and/or by stem-loop structures, while in Eukaryotes the cap-binding complex is responsible for this task. Here, we show that binding of translation initiation factor a/eIF2(γ) to the 5'-end of mRNA counteracts the 5'-to-3' exoribonucleolytic activity of Sso-RNase J in vitro. Hence, 5'-to-3' directional decay and 5'-end protection appear to be conserved features of mRNA turnover in all kingdoms of life.     

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.