M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

Assembly of Lampbrush Chromosomes from Sperm Chromatin

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24–48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific “landmarks.” Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.

Activation of p53 in cervical carcinoma cells by small molecules

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21WAF1/CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.

Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.

Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells.

Activation of Ca2+ signaling in neutrophils by the mast cell-released immunophilin FKBP12.

The immunophilins of the FK506-binding protein (FKBP) family are intracellular proteins that bind the immunosuppresants FK506 and rapamycin. In this study we show that HMC-1 mast cells sensitized with IgE release FKBP12 upon stimulation with anti-IgE. The release is rapid and not affected by actinomycin D or cycloheximide, suggesting that it is due to exocytosis from a storage compartment. FKBP12 from HMC-1 mast cells exhibits biological activity. When applied extracellularly to human neutrophils, it induces transient changes in the intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ release from intracellular stores. Inhibition of [Ca2+]i changes by ruthenium red and ryanodine indicates that ryanodine receptor/Ca2+ release channels are involved in FKBP12-induced Ca2+ signaling. Neutrophil activation by mast cell-derived FKBP12 is prevented by complexing FKBP12 with FK506 or rapamycin. These results demonstrate that extracellular FKBP12 functions as a cytokine in cell-to-cell communication. They further suggest a pathophysiological role for FKBP12 as a mediator in immediate or type I hypersensitivity and may have implications for novel therapeutic strategies in the treatment of allergic disorders with FK506 and rapamycin.