Summary RNA-mediated gene silencing in human cells requires the accurate generation of ∼22-nucleotide microRNAs (miRNAs) from double-stranded RNA substrates by the endonuclease Dicer. Although the phylogenetically conserved RNA-binding proteins TRBP and PACT are known to contribute to this process, their mode of Dicer binding and their genome-wide effects on miRNA processing have not been determined. We solved the crystal structure of a human Dicer–TRBP interaction complex comprising two domains of previously unknown structure. Interface residues conserved between TRBP and PACT show that the proteins bind to Dicer in a similar manner and by mutual exclusion. Based on the structure, a catalytically active Dicer that cannot bind TRBP or PACT was designed and introduced into Dicer-deficient mammalian cells, revealing selective defects in guide strand selection. These results demonstrate the role of Dicer-associated RNA binding proteins in maintenance of gene silencing fidelity.

The yeast two-hybrid system was used to identif proteins that interact with Ras. The H-Ras protein was found to interact with a g nnucleotide di ostimulator (GDS) that has been previously shown to regulate guanine nudeotide exchange on another member of the Ras protein family, Ral. The interaction is meted by the C-terminal, catalytic segment ofthe RalGDS and can be detected both in vivo, using the two-hybrid system, and in vto, with purfi recombinant proein. The inter of the RaIGDS C-teri segment with Ras is specific, dependent on activation ofRas by GTP, and blocked by a muttion that affects Ras effector fction. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, n that the RalGDS may also be a Ras effector. Consistent with this idea, the RaIGDS was found to inhibit the binding of Raf to Ras.The H-ras protooncogene plays a critical role in regulating cell growth, motility, and differentiation, and it is mutationally activated in many types ofcancer (1). The product ofthis gene is a membrane-associated protein that is the prototype of a family of small GTPases. This family includes the Rac and Rho proteins, which regulate cytoskeletal function; the Rab proteins, which are involved in intracellular vesicle transport; the Ran protein, which is involved in nuclear transport and cell cycle control; and the Ral proteins, which are membrane or vesicle-associated proteins of unknown function (2).Each of these proteins functions as a molecular switch, transmitting a signal in the active GTP-bound state and reverting to an inactive state when the bound GTP is hydrolyzed to GDP. The intrinsic GTPase activity ofthese proteins is accelerated by GTPase-activating proteins or GAPs, of which Ras-GAP (p1200AP) is the best characterized (3). The function of the small GTPases is positively regulated by guanine nucleotide exchange factors or dissociation stimulators (GDS proteins), which catalyze the exchange of GDP and GTP (3,4 Two-Hybrid Assays. The yeast reporter strain Y153 (MATa ura3-52 leu2-3,112 his3-200 ade2-101 trpl-901 gal4A gal80A LYS2::GALI-HIS3 GAL)::GALI-lacZ) (17) was used as host. Cells were transformed with Gal4 DNA-binding and activation domain fusion plasmids by using the lithium acetate procedure (18) and plated on selective synthetic medium (0.67% yeast nitrogen base/2% sucrose with appropriate Abbreviations: GAP, GTPase-activating protein; GDS, guanine nucleotide dissociation stimulator; GST, glutathione S-transferase; GMP-PCP, 5'-guanylyl methylenediphosphate.

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Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programmed cell death. The biochemical milieu regulates mitochondrial function and contributes to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of ageing. Extracellular matrix stiffness and cytoskeletal tension are also altered in cancer and in aged tissues. We 5 determined that cytoskeletal tension elicits a mitochondrial stress response that modifies mitochondrial function via SLC9A1-dependent ion exchange and HSF1-dependent transcription. Our data indicate that this cytoskeletal tension-induced mitochondrial stress response, termed mitohormesis, adaptively tunes mitochondrial metabolism and facilitates oxidative stress resilience. These findings demonstrate that cytoskeletal 10 tension regulates mitochondrial function and suggests that mechanical forces influence tissue behavior by modulating mitochondrial metabolism.One Sentence Summary: Adhesion-mediated mechanotransduction programs mitochondrial metabolism through an adaptive stress response.Main Text: Nutrient availability, oxygen content, and pH regulate cellular metabolism. 15Extracellular matrix (ECM) stiffness can change cellular metabolism by regulating the levels and/or activity of cytoplasmic enzymes responsive to cytoskeletal polymerization (1, 2). Many aspects of cellular metabolism depend upon the mitochondria, a key metabolic organelle that is structurally altered by physical forces and the cytoskeleton (3-5). Since mitochondrial structure influences mitochondrial function, we sought to 20 determine if ECM stiffness and cytoskeletal tension influence cellular metabolism by regulating mitochondrial structure and function. Cytoskeletal tension alters mitochondrial structure and functionTo explore associations between ECM stiffness and mitochondrial structure, we examined the mitochondrial morphology of nonmalignant human mammary epithelial 25 cells (MECs; MCF10A) cultured for 24 hours on polyacrylamide hydrogel gels (PA-gel) ranging in elasticity (stiffness) between normal breast stroma (400 Pa) and breast tumors (6k -60k Pa) (6, 7) (tissue culture polystyrene ~3G Pa). MECs cultured on this range of PA-gel elasticities displayed a variety of mitochondrial morphologies, ranging from thin interconnected filaments (400 Pa), to thickened filaments (6k Pa), and then ~300 nM 30 diameter fragments with toroidal shapes (60k Pa) ( Fig.1A and S1A-B). Cells respond to ECM stiffness by ligating ECM adhesion receptors that induce Rho-GTPase-dependent cytoskeletal remodeling and increase actomyosin tension through type-II myosins (8). Pharmacological inhibition of Rho-associated protein kinase (ROCK) with Y27632 or type-II myosins with blebbistatin, reduced the prevalence of the thick toroidal 35 mitochondrial fragments in MECs plated on the stiffest ECM PA-gels (Fig.1A). These data indicate that mitochondrial structure is sensitive to cytoskeletal tension generated in response to stiffness of the ext...