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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.The nucleolus serves many functions (4, 23, 36, 51); however, its most prominent function remains ribosome biogenesis. This process comprises rRNA gene transcription; processing of the 47S pre-rRNA to mature 18S, 5.8S, and 28S rRNAs; and assembly of preribosomal particles (21). Ribosome biogenesis is spatially organized in distinct compartments within the nucleolus. Transcriptionally active ribosomal genes are thought to be situated at either the boundaries of the fibrillar centers (FCs) and dense fibrillar components (DFCs) or in DFCs (45), whereas both the pre-rRNA processing and the assembly of preribosomal particles occur in the DFCs and granular components (GCs) (46). Ribosome biogenesis is regulated at multiple levels, including the transcription of ribosomal genes and the phosphorylation, methylation, and acetylation of component nucleolar factors, plus the trafficking and interaction of these factors (30).Proteasomal regulation has been implicated in many processes, including cell cycle progression, transcription, and antigen processing (24) (18,34,58). Indirect evidence has hinted at a possible role for the ubiquitin-proteasome system (UPS) in ribosomal biogenesis. Two different ubiquitination patterns have been reported for the late processing factor B23 (25,48). Several other ribosomal factors may be ubiquitinated, as suggested by a recent proteomic analysis in yeast (37). It has also been known for many years that two ubiquitin precursors are ribosomal fusion proteins (5,16,47). A large ribosomal subunit protein (L28) forms the most abundant ubiquitin-protein conjugate in yeast, and this modification is essential for ribosome function and efficient translation (53). A possible role for ubiquitin in nucleolus disassembly was also suggested (54). In addition, a ubiquitin ligase is known to regulate the processing and nuclear export of rRNA, as well as tRNA and mRNA in yeast (35). Finally, a temperature-sensitive point mutation of the Cic1p/Nsa3p yeast protein, an adaptor for the 26S proteasome, associates with early pre-60S particles (49), and inhibits both the synthesis of the mature 5.8S and 25S rRNAs and the releas...

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.

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 7alpha-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.

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