Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli

A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.     

Yeast and human RNA helicases involved in ribosome biogenesis: Current status and perspectives

Ribosome biogenesis is a fundamental process that is conserved in eukaryotes. Although spectacular progress has been made in understanding mammalian ribosome synthesis in recent years, by far, this process has still been best characterised in the yeast Saccharomyces cerevisiae. In yeast, besides the rRNAs, the ribosomal proteins and the 75 small nucleolar RNAs, more than 250 non-ribosomal proteins, generally referred to as trans-acting factors, are involved in ribosome biogenesis. These factors include nucleases, RNA modifying enzymes, ATPases, GTPases, kinases and RNA helicases. Altogether, they likely confer speed, accuracy and directionality to the ribosome synthesis process, however, the precise functions for most of them are still largely unknown. This review summarises our current knowledge on eukaryotic RNA helicases involved in ribosome biogenesis, particularly focusing on the most recent advances with respect to the molecular roles of these enzymes and their co-factors in yeast and human cells. This article is part of a Special Issue entitled: The Biology of RNA helicases—Modulation for life.     

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

Ribosomal RNA processing and ribosome biogenesis in eukaryotes

In eukaryotes nearly 500 rRNAs, ribosomal proteins, snoRNAs and trans-acting factors contribute to ribosome biogenesis. After more than 30 years of intense research, the incredible complexities of nucleolar function are revealed but details often remain unclear. Here we review this progress and the many intriguing questions which remain.     

DNA methyltransferase inhibition may limit cancer cell growth by disrupting ribosome biogenesis

Mutations in the pattern of CpG methylation imprinting of the human genome have been correlated with a number of diseases including cancer. In particular, aberrant imprinting of tumor suppressor genes by gain of CpG methylation has been observed in many cancers and thus represents an important alternative pathway to gene mutation and tumor progression. Inhibitors of DNA methylation display therapeutic effects in the treatment of certain cancers and it has been assumed that these effects are due to the reversal of mutant gene imprinting. However, significant reactivation of imprinted tumor suppressor genes is rarely observed in vivo following treatment with DNA methylation inhibitors. A recent study revealed an unexpected requirement for CpG methylation in the synthesis and assembly of the ribosome, an essential function for cell growth and proliferation. As such, the data provide an unforeseen explanation of the action of DNA methylation inhibitors in restricting cancer cell growth.Key words: DNA methylation, meCpG, DNA methyltransferase-inhibition, DNMT1^-/-, DNMT3b^-/-, aza-deoxycytidine, gene silencing, ribosome biogenesis, cancer therapy     

Regulated expression and intracellular localization of cystatin F in human U937 cells.

Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin ( approximately 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3-4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPalpha, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong ( approximately 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.     

Changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential

Many human pathological conditions, not linked to genetic alterations of oncogenes or tumor suppressors, are nevertheless associated with an increased risk of developing cancer, and some of them are characterized by quantitative and/or qualitative changes in ribosome biogenesis. Indeed, there is evidence that both an up-regulation of ribosome biogenesis, such as that occurring during the abnormal stimulation of cell growth, and intrinsic dysfunctions of ribosomes, such as those characterizing a series of inherited disorders, show an increased incidence of tumor onset. Here we discuss some recent insights into the mechanisms by which these alterations in ribosome biogenesis may facilitate tumorigenesis.     

Changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential

Many human pathological conditions, not linked to genetic alterations of oncogenes or tumor suppressors, are nevertheless associated with an increased risk of developing cancer, and some of them are characterized by quantitative and/or qualitative changes in ribosome biogenesis. Indeed, there is evidence that both an up-regulation of ribosome biogenesis, such as that occurring during the abnormal stimulation of cell growth, and intrinsic dysfunctions of ribosomes, such as those characterizing a series of inherited disorders, show an increased incidence of tumor onset. Here we discuss some recent insights into the mechanisms by which these alterations in ribosome biogenesis may facilitate tumorigenesis.     

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.     

A chemical strategy to manipulate the intracellular localization of drugs in resistant cancer cells

A number of multidrug-resistant (MDR) cancer cells have been shown to have acquired an increased capacity to sequester weakly basic anticancer drugs in their lysosomes relative to drug-sensitive counterparts. In this report we have comparatively evaluated the concentrations of the anticancer agent daunorubicin (DNR) in intracellular compartments of drug-sensitive and MDR HL-60 cell lines, both of which do not express common efflux transporters such as P-glycoprotein at the plasma membrane. Our results suggest that lysosomal sequestration plays a significant role in the emergence of MDR since it effectively limits the drug's ability to interact with target molecules located in the nucleus. Using a series of weakly basic structural isomers with variable basicity, we illustrate that the magnitude of the pKa value correlates with the degree of lysosomal sequestration. Accordingly, a series of structurally modified forms of DNR with reduced basicity were synthesized, and their intracellular distribution was evaluated. Consistent with model compounds, derivatives of DNR with lowered pKa values showed visibly reduced lysosomal sequestration in two separate MDR cell lines. Collectively, this work highlights the importance of understanding the intracellular localization of drugs and proposes a rational strategy to manipulate it.