The biochemistry of RNA metabolism studied in situ

In vitro assays have contributed important insights into the mechanisms of RNA metabolism in cells. A growing collection of microscopy techniques is allowing the measurement of macromolecular binding and complex formation in the context of a real cell. We will first discuss two of the more established techniques. Fluorescence resonance energy transfer (FRET) identifies binding partners, pairs of molecules residing in the same macromolecular complexes. The complimentary technique of fluorescence recovery after photobleaching (FRAP) measures the rates of binding and unbinding of those molecules in their complexes. A newer technique--in vitro FRAP--assesses the regulation of binding and complex formation by co-factors in the nucleus

Experimental observations of a nuclear matrix

Nuclei are intricately structured, and nuclear metabolism has an elaborate spatial organization. The architecture of the nucleus includes two overlapping and nucleic-acid-containing structures - chromatin and a nuclear matrix. The nuclear matrix is observed by microscopy in live, fixed and extracted cells. Its ultrastructure and composition show it to be, in large part, the ribonucleoprotein (RNP) network first seen in unfractionated cells more than 30 years ago. At that time, the discovery of this RNP structure explained surprising observations that RNA, packaged in proteins, is attached to an intranuclear, non-chromatin structure. Periodic and specific attachments of chromatin fibers to the nuclear matrix create the chromatin loop domains that can be directly observed by microscopy or inferred from biochemical experiments. The ultrastructure of the nuclear matrix is well characterized and consists of a nuclear lamina and an internal nuclear network of subassemblies linked together by highly structured fibers. These complex fibers are built on an underlying scaffolding of branched 10-nm filaments that connect to the nuclear lamina. The structural proteins of the nuclear lamina have been well characterized, but the structural biochemistry of the internal nuclear matrix has received less attention. Many internal matrix proteins have been identified, but far less is known about how these proteins assemble to make the fibers, filaments and other assemblies of the internal nuclear matrix. Correcting this imbalance will require the combined application of biochemistry and electron microscopy. The central problem in trying to define nuclear matrix structure is to identify the proteins that assemble into the 10-nm filaments upon which the interior architecture of the nucleus is constructed. Only by achieving a biochemical characterization of the nuclear matrix will we advance beyond simple microscopic observations of structure to a better understanding of nuclear matrix function, regulation and post-mitotic assembly

Mammalian SWI/SNF Enzymes and the Epigenetics of Tumor Cell Metabolic Reprogramming

Tumor cells reprogram their metabolism to survive and grow in a challenging microenvironment. Some of this reprogramming is performed by epigenetic mechanisms. Epigenetics is in turn affected by metabolism; chromatin modifying enzymes are dependent on substrates that are also key metabolic intermediates. We have shown that the chromatin remodeling enzyme Brahma-related gene 1 (BRG1), an epigenetic regulator, is necessary for rapid breast cancer cell proliferation. The mechanism for this requirement is the BRG1-dependent transcription of key lipogenic enzymes and regulators. Reduction in lipid synthesis lowers proliferation rates, which can be restored by palmitate supplementation. This work has established BRG1 as an attractive target for breast cancer therapy. Unlike genetic alterations, epigenetic mechanisms are reversible, promising gentler therapies without permanent off-target effects at distant sites

BRG1, a SWI/SNF chromatin remodeling enzyme ATPase, is required for maintenance of nuclear shape and integrity

We recently reported that reducing the levels of BRG1, the catalytic subunit of mammalian SWI/SNF chromatin remodeling enzymes, induces alterations in nuclear shape in a breast epithelial cell line. Immunostaining the BRG1 knockdown cells with nuclear lamina antibodies revealed a significantly increased frequency of grooves, or invaginations, in the nuclei. Disruption of each of the major cytoplasmic filament systems (actin, tubulin and cytokeratins) had no impact on the BRG1-dependent changes in nuclear shape, indicating that the observed changes in nuclear morphology are unlikely to be a result of alterations in the integrity of the nuclear-cytoplamic contacts in the cell. We propose that the BRG1-dependent nuclear shape changes reflect a role for the chromatin remodeling enzyme in maintaining the structural integrity of the nucleus via global regulation of chromatin structure and dynamics within the nucleus

Regulation of mRNA Export by the PI3 kinase / AKT Signal Transduction Pathway

UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5\u27 end of a nuclear mRNA, but are also reported to associate with the Exon Junction Complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly we used Fluorescence Recovery after Photobleaching (FRAP) to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the PI3 kinase / AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 Kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intron-less Histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the Endoplasmic Reticulum or to Mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and can promote the nuclear retention of some mRNAs

In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)-labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)-dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility

Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture

<p>Abstract</p> <p>Background</p> <p>MCF-10A cells are near diploid and normal human mammary epithelial cells. In three-dimensional reconstituted basement membrane culture, they undergo a well-defined program of proliferation, differentiation, and growth arrest, forming acinar structures that recapitulate many aspects of mammary architecture <it>in vivo</it>. The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line. This was followed by repeated selection for increasingly aggressive tumor formation from cells recovered from xenograft tumors in immuno-compromised mice, generating the MCF-10CA1a cell line. When inoculated subcutaneously into the flanks of immuno-compromised mice, MCF-10AT cells occasionally form tumors, whereas MCF-10CA1a cells invariably form tumors with a shorter latency than MCF-10AT derived tumors.</p> <p>Results</p> <p>MCF-10AT cells grown in three-dimensional basement membrane culture form complex multi-acinar structures that produce a basement membrane but undergo delayed cell cycle arrest and have incomplete luminal development. MCF-10CA1a cells grown in three-dimensional basement membrane culture form large, hyper-proliferative masses, that retain few characteristics of MCF10A acini and more closely resemble tumors.</p> <p>Conclusion</p> <p>Here we report on the growth and differentiation properties of these three matched cell lines in three-dimensional basement membrane culture. Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance. The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.</p

Binding of ATP to UAP56 is necessary for mRNA export

The major-histocompatibility-complex protein UAP56 (BAT1) is a DEAD-box helicase that is deposited on mRNA during splicing. UAP56 is retained on spliced mRNA in an exon junction complex (EJC) or, alternatively, with the TREX complex at the 5\u27 end, where it might facilitate the export of the spliced mRNA to the cytoplasm. Using confocal microscopy, UAP56 was found to be concentrated in RNA-splicing speckled domains of nuclei but was also enriched in adjacent nuclear regions, sites at which most mRNA transcription and splicing occur. At speckled domains, UAP56 was in complexes with the RNA-splicing and -export protein SRm160, and, as measured by FRAP, was in a dynamic binding equilibrium. The application of an in vitro FRAP assay, in which fluorescent nuclear proteins are photobleached in digitonin-extracted cells, revealed that the equilibrium binding of UAP56 in complexes at speckled domains was directly regulated by ATP binding. This was confirmed using a point mutant of UAP56 that did not bind ATP. Point mutation of UAP56 to eliminate ATP binding did not affect RNA splicing, but strongly inhibited the export of mRNA to the cytoplasm

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer