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Cell and Developmental Biology
Ph.D., 1985, Michigan State University
Nuclear Architecture and Gene Expression
Nucleic acid metabolism is architecturally organized in the eukaryotic nucleus. Nucleic acids themselves- as well as their metabolism in transcription, RNA processing, and RNA export- are structurally constrained to dynamic nuclear domains. Our larger goal is to understand the mechanisms that accomplish the self-assembly of these domains and achieve the spatial organization of gene expression. Our approach is an interdisciplinary one, combining biochemistry and molecular biology with confocal and electron microscopy.
One ongoing project studies the regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway. Two complexes forming on an mRNA may play a role in its export to the cytoplasm. The export proteins UAP56, ALY/REF, and NXF1-p15 sequentially bind to a TRanscription/EXport (TREX) complex, located at the 5’ end NXF1 recruited to the complex may subsequently bind at nuclear pores for export of the mRNA to the cytoplasm. A second complex, the Exon Junction Complex (EJC), with a core of eIF4A3, MLN51, MAGOH, and Y14, forms on the mRNA at sites 24 nucleotides upstream from exon-exon junctions. We used fluorescence recovery after photobleaching (FRAP) as a live cell screen to find signal transduction pathways that alter the binding of these proteins in mRNA complexes. Our results show that PI3 kinase/ AKT regulate the binding affinity of UAP56, NXF1, eIF4A3, MAGOH, and Y14 in mRNA-associated complexes in live cells. In addition, MAGOH binding is strongly reduced by the inhibition of mTORC1. The active PI3 Kinase/AKT pathway causes increased nuclear retention of a subset of mRNAs in the nucleus, consistent with this signal transduction pathway regulating the export of mRNA.
A second ongoing project, conducted as a long-term collaboration with the laboratory of Tony Imbalzano, also in this department, studies the role of chromatin remodeling in breast cancer. This project has shown that the SWI/SNF chromatin remodeling enzymes BRG1 and BRM are required for the proliferation of human mammary epithelial cells in conventional tissue culture and in three dimensional reconstituted basement membrane cultures where these cells develop into tissue-like acini. In addition, reduction in BRG1 levels causes nuclear shape changes that are independent of nuclear-cytoskeletal connections and are mediated by internal nuclear forces.
We have presented evidence that nuclear RNA and the ongoing synthesis of RNA are required for maintaining the normal architecture of the nucleus and have proposed that long non-coding RNAs play this structural role. A goal for future work is to test this hypothesis and identify specific architectural RNAs in the nucleus.
Director for Cell Biology Confocal Core - Three Dimensional Microscopy - www.umassmed.edu/3dml
Figure: Resinless section of a CaSki cell nuclear matrix. Solubleproteins and chromatin have been removed from this nucleus, uncovering thenuclear matrix which consists of two parts. The nuclear lamina isthe outer shell of the matrix which lies just under the nuclear envelopeand is primarily composed to the lamin proteins A, B, and C. Connected tothe lamina and extending throughout the nuclear volume is the internalnuclear matrix, an intricate structure built on a scaffolding of 10nm filaments whose molecular composition remains unknown. The largest massesremaining in the interior are remnants of nucleoli.