Small RNAs direct RNA induced silencing complexes (RISCs) to regulate the stability and translation of mRNAs1,2. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies3. However, RISC proteins can associate with membrane compartments such as the Golgi and ER4. Here, we show that GW-bodies are associated with late endosomes or multivesicular bodies (MVBs). Blocking turnover of MVBs into lysosomes by loss of the tethering factor HPS45, enhances siRNA- and miRNA-mediated silencing in Drosophila and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT6 depletion results in impaired miRNA silencing and loss of GW-bodies in cells. These results indicate that active RISC is physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISC to load small RNAs. We suggest that recycling of RISC is promoted by MVBs in order to more effectively engage with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.

Highlights d SRPK1 phosphorylates protamine to initiate paternal genome reprogramming d Phosphorylation site mutations in protamine inhibit protamine-to-histone exchange d Phosphorylated protamine permits efficient recruitment of NPM2 and HIRA d Paternal and maternal genomes undergo synchronized reprogramming upon fertilization

a b s t r a c tSmall non-coding RNAs regulate gene expression in a sequence-specific manner. In Drosophila, Dicer-2 (Dcr-2) functions in the biogenesis of endogenous small interfering RNAs (endo-siRNAs). We identified 21 distinct proteins that exhibited a P1.5-fold change as a consequence of loss of dcr-2 function. Most of these were metabolic genes implicated in stress resistance and aging. dcr-2 Mutants had reduced lifespan and were hypersensitive to oxidative, endoplasmic reticulum, starvation, and cold stresses. Furthermore, loss of dcr-2 function led to abnormal lipid and carbohydrate metabolism. Our results suggest roles for the endo-siRNA pathway in metabolic regulation and defense against stress and aging in Drosophila.

The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein ( Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.-Lim, D.-H., Lee, S., Han, J. Y., Choi, M.-S., Hong, J.-S., Seong, Y., Kwon, Y.-S., Lee, Y. S. Ecdysone-responsive microR-252-5p controls the cell cycle by targeting Abi in Drosophila.

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.

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