Summary Background Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, mis-orientation creates multiple lumens. Results We now show that silencing of the polarity protein Par3 causes spindle mis-orientation in MDCK cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically-tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3, or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. Conclusions The Par3/aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation.

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development.

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3 untranslated regions that act as mRNA stability determinants by interacting with ARE-binding proteins (ARE-BPs). We have destabilized two mRNAs by fusing sequence-specific RNA-binding proteins to KSRP, a decay-promoting ARE-BP, in a tethering assay. These results support a model that KSRP recruits mRNA decay machinery/factors to elicit decay. The ability of tethered KSRP to elicit mRNA decay depends on functions of known mRNA decay enzymes. By targeting the Rev response element of human immunodeficiency virus type 1 by using Rev-KSRP fusion protein, we degraded viral mRNA, resulting in a dramatic reduction of viral replication. These results provide a foundation for the development of novel therapeutic strategies to inhibit specific gene expression in patients with acquired or hereditary diseases.mRNA stability varies considerably from one mRNA species to another and plays an important role in determining levels of gene expression (19,46,47). Differential mRNA decay rates are determined by specific cis-acting elements within the mRNA molecule. The most common cis element responsible for rapid mRNA decay in mammalian cells is the AU-rich element (ARE) present within the 3Ј untranslated regions (UTRs) of a variety of short-lived mRNAs (2, 10). Recent computational analysis of the 3Ј UTRs revealed that as many as 8% of human mRNAs contain AREs (2). This finding suggests that AREs may account for degradation of most unstable mRNAs in human cells.

Regulated mRNA decay is a highly important process for the tight control of gene expression. Inherently unstable mRNAs contain AU-rich elements (AREs) in the 39 untranslated regions that direct rapid mRNA decay by interaction with decay-promoting AREbinding proteins (ARE-BPs). The decay of ARE-containing mRNAs is regulated by signaling pathways that are believed to directly target ARE-BPs. Here, we show that BRF1 involved in ARE-mediated mRNA decay (AMD) is phosphorylated by MAPK-activated protein kinase 2 (MK2). In vitro kinase assays using different BRF1 fragments suggest that MK2 phosphorylates BRF1 at four distinct sites, S54, S92, S203, and an unidentified site at the C terminus. Coexpression of an active form of MK2 inhibits ARE mRNA decay activity of BRF1. MK2-mediated inhibition of BRF1 requires phosphorylation at S54, S92, and S203. Phosphorylation of BRF1 by MK2 does not appear to alter its ability to interact with AREs or to associate with mRNA decay enzymes. Thus, MK2 inhibits BRF1-dependent AMD through direct phosphorylation. Although the mechanism underlying this inhibition is still unclear, it appears to target BRF1-dependent AMD at a level downstream from RNA binding and the recruitment of mRNA decay enzymes.

Background: Nuclear membrane protein emerin binding to nuclear intermediate filaments (lamins) and BAF contributes to forming a nuclear "lamina" structure. Results: Emerin is O-GlcNAc-modified at eight sites: two (Ser-53 and Ser-54) influence further O-GlcNAcylation, and one (Ser-173) regulates association with BAF in the chromatin/lamin B "niche." Conclusion: O-GlcNAc transferase, a nutrient-responsive enzyme, regulates emerin. Significance: Emerin hyper-O-GlcNAcylation may contribute to cardiomyopathy and other conditions.