Wataru Komatsu

Potential roles for ubiquitin and the proteasome during ribosome biogenesis.

ABSTRACT We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.

Suppression of hypercholesterolemia in hepatoma-bearing rats by cabbage extract and its component, S-methyl-L-cysteine sulfoxide.

The effect of cabbage extract on cholesterol metabolism was studied in Donryu rats subcutaneously implanted with an ascites hepatoma cell line (AH109A). The hepatoma-bearing rats exhibited hypercholesterolemia induced by increasing cholesterogenesis in the host liver and decreasing steroid excretion into feces. The cabbage extract intake or administration reduced serum cholesterol level and enhanced fecal bile acid excretion and cholesterol 7α-hydroxylase activity, the rate-limiting enzyme of bile acid biosynthesis, in the microsomal fraction of the liver. Furthermore, S-methyl-l-cysteine sulfoxide, a component of cabbage, could mimic the effect of cabbage extract when orally administered. These results suggest that cabbage suppresses hypercholesterolemia responding to hepatoma growth by upregulating cholesterol catabolism and that S-methyl-l-cysteine sulfoxide in cabbage is one of the factors suppressing hypercholesterolemia in the hepatoma-bearing rats.

Association of human DNA helicase RecQ5β with RNA polymerase II and its possible role in transcription

Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.

Splicing Factor 2-Associated Protein p32 Participates in Ribosome Biogenesis by Regulating the Binding of Nop52 and Fibrillarin to Preribosome Particles

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52.

Modification of tumor necrosis factor and interleukin-1 productivity in macrophages from hepatoma-bearing rats by dietary proteins

Abstract Changes in tumor necrosis factor (TNF) and interleukin-1 (IL-1) productivity in resident peritoneal macrophages and those in the serum lipid levels were traced for up to 14 days after subcutaneous implantation of hepatoma (AH109A) cells to rats kept on a 20% casein diet (20C). Elevated levels of serum triglyceride and cholesterol were found to be associated with growth of the solid hepatoma. The ability of macrophages to produce TNF and IL-1 rose twice at both the early (days 2–4) and late (day 14) stages. To examine the effects of dietary proteins, hepatoma-free (normal) and -bearing rats were maintained on the 20C or a 20% gluten diet (20G) for 14 days after sham or hepatoma implantation. No significant changes were noticed in the serum lipid levels between the 20C and 20G groups in either hepatoma-free or -bearing rats. TNF and IL-1 production by macrophages from hepatoma-bearing rats was significantly higher in the 20C group than in the 20G group. These results indicate that TNF and IL-1 productivity in macrophages is enhanced by hepatoma implantation and that their productivity may be modified by dietary proteins in the hepatoma-bearing state.

Human cell growth regulator Ly-1 antibody reactive homologue accelerates processing of preribosomal RNA.

Ribosome biogenesis is an essential process for cell growth and proliferation and is enhanced in cancer and embryonic stem cells. Mouse Ly‐1 antibody reactive clone product (Lyar) is expressed at very high levels in many tumor, leukemia or embryonic stem cells; is a novel nucleolar protein with zinc‐finger DNA‐binding motifs and is involved in cell growth regulation. However, cellular function of Lyar remains unexplored. Here, we show that human homologue of Lyar (LYAR) accelerates ribosome biogenesis at the level of processing of preribosomal RNA (pre‐rRNA). We show that LYAR is excluded from the nucleolus after actinomycin D treatment and is present in preribosomal fraction of the nuclear extract as well as in the fractions with 40S, 60S and 90S sedimentation coefficients. LYAR is required for processing of 47S/45S, 32S, 30S and 21S pre‐rRNAs. In addition, we show that over‐expression of LYAR increases cell proliferation without affecting the expression of c‐Myc or p53. Combined, these results suggest that some rapidly growing cells enhance ribosome biogenesis by increasing the expression of LYAR.

Induction of Tumor Necrosis Factor Production and Antitumor Effect by Cabbage Extract

The effect of cabbage extract on the production of tumor necrosis factor and its implication in the antitumor effect were examined in vitro and in vivo. Cabbage extract stimulated the production of tumor necrosis factor by rat spleen cells and showed cytotoxic activity in a rat ascites hepatoma cell line (AH109A) when hepatoma cells were cultured with cabbage-stimulated spleen cells. When the extract was administered orally to AH109A-bearing rats in combination with lipopolysaccharide injection, the hepatoma weights were reduced to one-half of the vehicle control. The cytotoxic activity of tumor-infiltrating macrophages was induced by simultaneous treatment with cabbage extract and lipopolysaccharide. These results indicate that cabbage extract contains macrophage-stimulating component(s) and can implement the antitumor effect by stimulating the cytotoxicity of tumor-infiltrating macrophages.

Restoration by dietary glutamine of reduced tumor necrosis factor production in a low-protein-diet-fed rat model

Tumor necrosis factor-α (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.

In Vitro Stimulation of Tumor Necrosis Factor Production from Rat Splenocytes by Cabbage Extract

The effect of cabbage extract on in vitro production of tumor necrosis factor (TNF) was studied in primarily isolated rat splenocytes and peritoneal macrophages. Cabbage extract was found to stimulate the production of TNF in the cultured splenocytes, but not to do so in the cultured peritoneal macrophages. The production of TNF from peritoneal macrophages was significantly elevated by cabbage extract when these macrophages were co-cultured with isolated splenic T cells. The cytotoxic activities of the splenocytesstimulated with cabbageextract were significantly higher than those with vehicle control when the splenocytes were co-cultured with an ascites hepatoma cell line of AH109A. These results suggest that the stimulatory effect of cabbage extract on TNF production is indirect on macrophages and that T cells are involved in the appearance of the cabbageextract. They also suggest that in vitro anti-tumor activity is potentiated by cabbage extract through the stimulation of immune response.