UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex.

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported.

The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2.

Sphingolipid-based drugs selectively kill cancer cells by down-regulating nutrient transporter proteins.

Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable.

Differential effects of TBC1D15 and mammalian Vps39 on Rab7 activation state, lysosomal morphology, and growth factor dependence.

The small GTPase Rab7 promotes fusion events between late endosomes and lysosomes. Rab7 activity is regulated by extrinsic signals, most likely via effects on its guanine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP). Based on their homology to the yeast proteins that regulate the Ypt7 GTP binding state, TBC1D15, and mammalian Vps39 (mVps39) have been suggested to function as the Rab7 GAP and GEF, respectively.

Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis.

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7.

Ceramide-induced starvation triggers homeostatic autophagy.

Autophagy is triggered by ceramide, a sphingolipid that regulates diverse cellular processes including survival, differentiation and senescence. Both ceramide and autophagy play important, but incompletely understood, roles in type 2 diabetes and cancer. We reasoned that defining the connection between ceramide and autophagy might provide an important insight into these highly prevalent diseases.

Ceramide starves cells to death by downregulating nutrient transporter proteins.

Ceramide induces cell death in response to many stimuli. Its mechanism of action, however, is not completely understood. Ceramide induces autophagy in mammalian cells maintained in rich media and nutrient permease downregulation in yeast.

Transfection protocol for antisense oligonucleotides affects uniformity of transfection in cell culture and efficiency of mRNA target reduction.

In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variable transfection efficiency depending on the concentration of oligonucleotide used. A significant population of cells, especially at lower doses of oligonucleotide and cationic lipid, were untransfected.

Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs.

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression.

MicroRNA-143 regulates adipocyte differentiation.

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated.