MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC. A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358-AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope.

Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE). Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2). Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers.

Atypical PKC (aPKC) family members are involved in regulation of diverse cellular processes, including cell polarization. aPKCs are known to be activated by phosphorylation of specific threonine residues in the activation loop and turn motif. They can also be stimulated by interaction with Cdc42~GTP-Par6 complex. Here we report that PKCζ, a member of the aPKC family, is activated by SUMOylation. We show that aPKC is endogenously modified by SUMO1 and the nucleoporin Nup358 acts as its SUMO E3 ligase. Results from in vitro SUMOylation and kinase assays showed that the modification enhances the kinase activity of PKCζ by ~10-fold. By monitoring the phosphorylation of Lethal giant larvae (Lgl), a downstream target of aPKC, we confirmed these findings in vivo. Consistent with the function of Nup358 as a SUMO E3 ligase for aPKC, depletion of Nup358 attenuated the extent of SUMOylation and the activity of aPKC. Moreover, overexpression of the C-terminal fragment of Nup358 that possesses the E3 ligase activity enhanced SUMOylation of endogenous aPKC and its kinase activity. Collectively, our studies reveal a role for Nup358-dependent SUMOylation in the regulation of aPKC activity and provide a framework for understanding the role of Nup358 in cell polarity.Development and growth of an organism depend on the ability of cells to respond to extrinsic and intrinsic stimuli through distinct signaling cascades. Kinases are shown to be the major class of proteins involved in the relay of these signals The aPKC sub-family proteins have been well known for their role in generation and maintenance of polarity by their asymmetric localization within the cell 4 . Broadly, there are three well characterized polarity complexes, namely, the Par3-Par6-aPKC complex, Scribble-Dlg-Lgl and the Crumbs-PALS complex 5,6 . aPKC is a component of the Par polarity complex and is central to its functionality. Downstream to Par complex, aPKC is known to phosphorylate and regulate a range of polarity proteins, namely, Lgl, Par3, Crumbs, GSK3β , MARCKS, LGN and Lin5/NuMA 7 . Phosphorylation can affect the target protein in various ways; for example, phosphorylation of Par3 by aPKC causes it to dissociate from the Par3-Par6-aPKC complex, while phosphorylation of Lgl prevents it from localizing to the apical membrane 8 . Two activation modes of aPKC involving phosphorylation are known; one by phosphoinositide-dependent kinase-1 (PDK1)-mediated phosphorylation of specific threonine residue (T410 in PKCζ and T408 in PKCλ ) in the activation loop (A-loop) 9 and the other by mTORC2-directed phosphorylation of T560 in the turn motif (TM) of PKCζ 10 . Additionally, aPKC can be activated in a spatio-temporal manner by binding with the Cdc42~GTP-Par6 complex through its PB1 domain 11,12 . Interestingly, it is found that during epithelial polarization and asymmetric division of neural stem cells, aPKC makes mutually exclusive complexes with Par3 and Lgl [13][14][15] . This appears to be important for defining the membrane domains required for generation and...

The Par polarity complex, consisting of Par3, Par6 and atypical protein kinase C (aPKC), plays a crucial role in the establishment and maintenance of cell polarity. Although activation of aPKC is critical for polarity, how this is achieved is unclear. The developing zebrafish epidermis, along with its apical actin-based projections, called microridges, offers a genetically tractable system for unraveling the mechanisms of the cell polarity control. The zebrafish aPKC regulates elongation of microridges by controlling levels of apical Lgl, which acts as a pro-elongation factor. Here, we show that the nucleoporin Nup358 (also known as RanBP2)a component of the nuclear pore complex and a part of cytoplasmic annulate lamellae (AL)-SUMOylates zebrafish aPKC. Nup358-mediated SUMOylation controls aPKC activity to regulate Lgl-dependent microridge elongation. Our data further suggest that cytoplasmic AL structures are the possible site for Nup358-mediated aPKC SUMOylation. We have unraveled a hitherto unappreciated contribution of Nup358mediated aPKC SUMOylation in cell polarity regulation. This article has an associated First Person interview with the first author of the paper.