In the metazoan germline, piwi proteins and associated piwi-interacting RNAs (piRNAs) provide a defense system against the expression of transposable elements. In the cytoplasm, piRNA sequences guide piwi complexes to destroy complementary transposon transcripts by endonucleolytic cleavage. However, some piwi family members are nuclear, raising the possibility of alternative pathways for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromosomes. Knockdown of Piwi in the germline increases expression of transposable elements that are targeted by piRNAs, whereas protein-coding genes remain largely unaffected. Derepression of transposons upon Piwi depletion correlates with increased occupancy of Pol II on their promoters. Expression of piRNAs that target a reporter construct results in a decrease in Pol II occupancy and an increase in repressive H3K9me3 marks and heterochromatin protein 1 (HP1) on the reporter locus. Our results indicate that Piwi identifies targets complementary to the associated piRNA and induces transcriptional repression by establishing a repressive chromatin state when correct targets are found. Diverse small RNA pathways function in all kingdoms of life, from bacteria to higher eukaryotes. In eukaryotes, several classes of small RNA associate with members of the Argonaute protein family, forming effector complexes in which the RNA provides target recognition by sequence complementarity, and the Argonaute provides the repressive function. Argonaute-small RNA complexes have been shown to regulate gene expression both transcriptionally and post-transcriptionally. Post-transcriptional repression involves cleavage of target RNA through either the endonucleolytic activity of Argonautes or sequestering targets into cytoplasmic ribonucleoprotein (RNP) granules (Hutvagner and Simard 2008).

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

In Drosophila, two Piwi proteins, Aubergine (Aub) and Argonaute-3 (Ago3) localize to perinuclear ‘nuage’ granules and use guide piRNAs to target and destroy transposable element transcripts. We find that Aub and Ago3 are recruited to nuage by two different mechanisms. Aub requires a piRNA guide for nuage recruitment, indicating that its localization depends on recognition of RNA targets. Ago3 is recruited to nuage independently of a piRNA cargo and relies on interaction with Krimper, a stable component of nuage that is able to aggregate in the absence of other nuage proteins. We show that Krimper interacts directly with Aub and Ago3 to coordinate the assembly of the ping-pong piRNA processing (4P) complex. Symmetrical dimethylated arginines are required for Aub to interact with Krimper, but are dispensable for Ago3 to bind Krimper. Our study reveals a multi-step process responsible for the assembly and function of nuage complexes in piRNA-guided transposon repression.

The spatial and temporal regulation of the interactions among the ϳ60 proteins required for endocytosis is under active investigation in many laboratories. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (⌬⌬⌬⌬) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37°C. Live-cell imaging revealed that the dynamics of the early-and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor asparagine-prolinephenylalanine motifs binding to scaffold Eps15 homology domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis. INTRODUCTIONClathrin-mediated endocytosis (CME) mediates the internalization of extracellular molecules, plasma membrane lipids, and specific membrane proteins into the cell. Nutrient uptake, membrane remodeling, immune surveillance, and synaptic vesicle recycling all depend on CME to maintain and regulate many of their constituents (Geli and Riezman, 1998;Evans and Owen, 2002;Sorkin, 2004;Szymkiewicz et al., 2004). Thus, CME is an essential process for homeostasis and signaling regulation in all eukaryotic cells. CME involves Ͼ60 cytosolic proteins acting in concert to form a cargo-containing clathrin-coated vesicle (CCV). Current studies focus on elucidating the regulation and mechanisms of spatiotemporal coupling of endocytic proteins, beginning with the selection of cargo proteins at the plasma membrane by clathrin adaptors (Kaksonen et al., 2003(Kaksonen et al., , 2005Newpher et al., 2005). Adaptors recognize sorting signals in the cytoplasmic tail of cargo proteins, such as linear peptide motifs or posttranslational modifications such as ubiquitination (Wendland, 2002;Traub, 2003;Maldonado-Baez and Wendland, 2006). Adaptors also recruit clathrin and other endocytic proteins to sites of endocytosis (Wendland, 2002;Traub, 2003;Owen, 2004). Forming stable cargo-adaptorclathrin complexes at the plasma membrane is a key, ratelimiting early event in the assembly of CCVs (Ehrlich et al., 2004;Kaksonen et al., 2005;Newpher et al., 2005). Current models predict that these early endocytic complexes trigger the transition from early (cargo-gathering) to late (vesicle scission) events in endocytic internalization, although the mechanism is unknown (Ehrlich et al., 2004;Kaksonen et al., 2005;Merrifield et al., 2005;Naslavsky and Caplan, 2005;Newpher et al., 2005;Newpher and Lemmon, 2006).Most known adaptors are conserved from yeast to humans (Toret and Drubin, 2007); thus, we used the budding yeast Saccharomyces cerevisiae for this study...