Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development. We previously described the purification of a poly(A)-specific 3Ј-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue. Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported. Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein. The amino acid sequence of DAN shows homology to the RNase D family of 3Ј-exonucleases. DAN appears to be localized in both the nucleus and the cytoplasm. It is not stably associated with polysomes or ribosomal subunits. Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN. Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation. Ectopic expression of human DAN in enucleated oocytes rescues maturationspecific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.

is also initiated by deadenylation (Wilson and Treisman, D-35392 Giessen, Germany and 3 Department of Biology, University of 1988; Shyu et al., 1991; Chen and Shyu, 1995 1996). During oocyte maturation, deadenylation does not are not detectable in the enzyme preparation, and require specific cis-elements and is a default pathway PARN itself binds to m 7 GTP-Sepharose and is eluted for mRNAs that do not undergo compensatory poly(A) specifically with the cap analog m 7 GTP. Xenopus PARN elongation (Fox and Wickens, 1990; Varnum and is known to catalyze mRNA deadenylation during Wormington, 1990). In contrast, certain mRNAs which oocyte maturation. The enzyme is depleted from oocyte are polyadenylated during meiotic maturation contain extract with m 7 GTP-Sepharose, can be photocross-3Ј-UTR elements that promote their subsequent deadenyllinked to the m 7 GpppG cap and deadenylates ation after fertilization. Thus, the translation of these m 7 GpppG-capped RNAs more efficiently than ApppGmRNAs is restricted to mature oocytes (Bouvet et al., capped RNAs both in vitro and in vivo. These data 1994; Legagneux et al., 1995). In both cases, deadenylation provide additional evidence that PARN is responsible does not destabilize mRNAs immediately, but is a prefor deadenylation during oocyte maturation and sugrequisite for their degradation at later stages of developgest that interactions between 5Ј cap and 3Ј poly(A) ment (Audic et al., 1997; Gillian-Daniel et al., 1998; tail may integrate translational efficiency with mRNA Voeltz and Steitz, 1998). The uncoupling of deadenylation stability.from mRNA decay in gametes and embryos contrasts with Keywords: cap structure/deadenylation/mRNA stability/ both yeast and metazoan cells in which poly(A) removal oocyte maturation/poly(A) tails rapidly promotes mRNA degradation. Both the dependence of decapping on prior deadenylation and the inhibition of translation initiation by poly(A)

Cancers constitutively produce and secrete into the blood and other biofluids 30-150 nm-sized endosomal vehicles called exosomes. Cancer-derived exosomes exhibit powerful influence on a variety of biological mechanisms to the benefit of the tumors that produce them. We studied the immunosuppressive ability of epithelial ovarian cancer (EOC) exosomes on two cytotoxic pathways of importance for anticancer immunity-the NKG2D receptor-ligand pathway and the DNAM-1-PVR/nectin-2 pathway. Using exosomes, isolated from EOC tumor explant and EOC cell-line culture supernatants, and ascitic fluid from EOC patients, we studied the expression of NKG2D and DNAM-1 ligands on EOC exosomes and their ability to downregulate the cognate receptors. Our results show that EOC exosomes differentially and constitutively express NKG2D ligands from both MICA/B and ULBP families on their surface, while DNAM-1 ligands are more seldom expressed and not associated with the exosomal membrane surface. Consequently, the NKG2D ligand-bearing EOC exosomes significantly downregulated the NKG2D receptor expression on peripheral blood mononuclear cells (PBMC) while the DNAM-1 receptor was unaffected. The downregulation of NKG2D receptor expression was coupled to inhibition of NKG2D receptor-ligand-mediated degranulation and cytotoxicity measured in vitro with OVCAR-3 and K562 cells as targets. The EOC exosomes acted as a decoy impairing the NKG2D mediated cytotoxicity while the DNAM-1 receptor-ligand system remained unchanged. Taken together, our results support and explain the mechanism behind the recently reported finding that in EOC, NK-cell recognition and killing of tumor cells was mainly dependent on DNAM-1 signaling while the contribution of the NKG2D receptor-ligand pathway was complementary and uncertain.

Exosomes are specialized, nanometer-sized extracellular vesicles of endosomal origin actively secreted into the extracellular space by a variety of cells under normal and pathological conditions. Exosomes have recently emerged as important intercellular communicators and modulators of diverse mechanisms and cellular responses. Characterization of their composition and function will open possibilities for new diagnostic methods and promising therapeutic approaches based on nanobiology. This unit provides a standard isolation procedure for purification of exosomes based on density gradient ultracentrifugation with sucrose. The process of isolating exosomes relies on obtaining proper source fluids/supernatants as well as qualitative and quantitative assessment of the isolated vesicles. The methodological procedures here can be divided in three parts: (1) pre-isolation procedures aiming to obtain fluids containing exosomes, with a focus on protocols for organ explants and cell cultures; (2) a procedure for exosome isolation with several gradient alternatives; and (3) post-isolation procedures for estimating the purity and yield of the exosomal fraction. © 2016 by John Wiley & Sons, Inc.