Effect of ON 01910.Na, an anticancer mitotic inhibitor, on cell-cycle progression correlates with RanGAP1 hyperphosphorylation.

The benzyl styryl sulfone, ON 01910.Na, is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell lines. We examined the effect of ON 01910.Na on DNA damage-signaling molecules upstream of Cdc25C (Chk1, Chk2, and H2AX), as well as on Ran GTPase-activating protein 1 conjugated to small ubiquitin-related modifier 1 (RanGAP1·SUMO1), a mitosis coordinator. Prostate cancer, lymphoma, and leukemic cells were incubated with the drug for 4, 16, or 24 hours. Cell lysates were resolved on SDS-PAGE and analyzed by Western blot. Camptothecin and doxorubicin treatment caused activation/phosphorylation of DNA damage-responsive molecules by 4 hours, whereas ON 01910.Na did not do so. ON 01910.Na caused hyperphosphorylation of RanGAP1·SUMO1 within 4 hours that was sustained for more than 24 hours. Mild phosphorylation of Chk2 was observed only after 24-hour exposure, indicating that DNA damage response was not an initial effect of ON 01910.Na. MOLT-3 cells, synchronized by double-thymidine block, when released into a medium containing ON 01910.Na, accumulated mitotic cell number with a peak from 10 to 14 hours and remained near plateau for 20 hours, which corresponded with the time of RanGAP phosphorylation. ON 01910.Na had minimal effects on tubulin polymerization. These findings imply that ON 01910.Na neither induces DNA damage directly nor acts as a tubulin toxin. Its biological activity appears to rely on prolonged phosphorylation/hyperphosphorylation of RanGAP1·SUMO1. M-phase arrest and the consequent induction of apoptosis that follows could possibly be attributed to it. ON 01910.Na may act as an inhibitor of a RanGAP1·SUMO1 phosphatase or a stimulant of a new kinase. RanGAP1·SUMO1 appears to be a new target pathway for cancer chemotherapy.

Analysis of mRNA decay in Bacillus subtilis.

Studies of mRNA turnover in B. subtilis are less well known than in E. coli. Here we provide researchers who have an interest in gram-positive RNA processing with several protocols for RNA isolation, for 5'- and 3'-mapping of mRNAs and mRNA decay fragments, and we also include a comprehensive listing of B. subtilis mutants that are deficient in ribonucleases thought to be involved in mRNA decay.

Assay of Bacillus subtilis ribonucleases in vitro.

Significant progress has been made recently regarding the identification of the ribonucleases involved in RNA maturation and degradation in Bacillus subtilis. More than half of these enzymes have no ortholog in Escherichia coli. To confirm that the in vivo effects of mutations in genes encoding RNases are direct, it is often necessary to purify the enzymes and assay their activity in vitro. Development of such assays is also necessary for detailed biochemical analysis of enzyme properties. In this chapter, we describe the purification and assay of 12 RNases of B. subtilis thought to be involved in stable RNA maturation or RNA degradation.

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis.

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.

Participation of 3'-to-5' exoribonucleases in the turnover of Bacillus subtilis mRNA.

Four 3'-to-5' exoribonucleases have been identified in Bacillus subtilis: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5' ends were at the transcriptional start site and whose 3' ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of rpsO mRNA decay showed that decay intermediates accumulated as the result of a block to 3'-to-5' processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA trans translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.

Bacillus subtilis YhcR, a high-molecular-weight, nonspecific endonuclease with a unique domain structure.

In a continuing effort to identify ribonucleases that may be involved in mRNA decay in Bacillus subtilis, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3'-to-5' exoribonucleases (polynucleotide phosphorylase [PNPase], RNase R, and YhaM) was undertaken. These experiments revealed the presence of a high-molecular-weight nuclease encoded by the yhcR gene that was active in the presence of Ca(2+) and Mn(2+). YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3'-monophosphate products, similar to the well-characterized micrococcal nuclease of Staphylococcus aureus. YhcR appears to be located principally in the cell wall and is likely to be a substrate for a B. subtilis sortase. Zymogram analysis suggests that YhcR is the major Ca(2+)-activated nuclease of B. subtilis. In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named "NYD," for "new YhcR domain."

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.