Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin β. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H ؉ -ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H ؉ -ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics. INTRODUCTIONCellular compartments are very dynamic structures that adapt their shape, size, and number to cellular metabolism, differentiation state, and environmental conditions. Changes in mitochondrial shape during animal development (Chan, 2006), proliferation and degradation of peroxisomes in response to nutrient availability (Yan et al., 2005), and extensive fragmentation of the mammalian Golgi during mitosis (Shorter and Warren, 2002) are only a few examples highlighting organellar dynamics. Organelle homeostasis in the face of permanent membrane remodeling requires an equilibration and coordination of the fundamental processes of membrane fission and fusion.Important insights into membrane dynamics have come from studies of the vacuole of the budding yeast Saccharomyces cerevisiae. Vacuoles are highly dynamic structures that undergo regulated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to changing environmental conditions. When a yeast cell buds and initiates growth of a daughter cell, the vacuole pinches off vesicles that migrate into the growing daughter cell where they fuse to form the new vacuolar compartment (Weisman, 2003). Vacuolar rearrangements also occur when yeast cells are faced with nutrient limitation or osmotic stress. Exposure of yeast cells to hypertonic medium induces fragmentation of the vacuole into numerous small vacuolar vesicles, a process involving Fab1p-mediated phosphatidylinositol-3,5-bisphosphate synthesis (Efe et al., 2005). Conversely, in fast adaptation to hypotonic shock, vacuoles fuse to give rise to a more voluminous vacuole. These rapid change...

Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.

In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5'-cap, 5'- and 3'-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.

The survival motor neuron (SMN) complex functions in maturation of uridine-rich small nuclear ribonucleoprotein (RNP) particles. SMN mediates the cytoplasmic assembly of Sm proteins onto uridine-rich small RNAs, and then participates in targeting RNPs to nuclear Cajal bodies (CBs). Recent studies have suggested that phosphorylation might control localization and function of the SMN complex. Here, we show that the nuclear phosphatase PPM1G/PP2Cγ interacts with and dephosphorylates the SMN complex. Small interfering RNA knockdown of PPM1G leads to an altered phosphorylation pattern of SMN and Gemin3, loss of SMN from CBs, and reduced stability of SMN. Accumulation in CBs is restored upon overexpression of catalytically active, but not that of inactive, PPM1G. This demonstrates that PPM1G's phosphatase activity is necessary to maintain SMN subcellular distribution. Concomitant knockdown of unr interacting protein (unrip), a component implicated in cytoplasmic retention of the SMN complex, also rescues the localization defects. Our data suggest that an interplay between PPM1G and unrip determine compartment-specific phosphorylation patterns, localization, and function of the SMN complex.