We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFN␥ signaling and type II IFN responses.IFNs are cytokines that exhibit antiviral, immunomodulatory, growth-inhibitory, and cytotoxic properties (1-12). The critical roles of these cytokines in the innate immune system have provoked clinical interest and extensive studies to explore their therapeutic potential. These studies, spanning several decades, have definitively established their utility in the treatment of viral syndromes, many malignancies, and some autoimmune disorders (1-12).IFN␥, the sole type II IFN, binds to the IFNGR1 and IFNGR2 subunits of the type II IFN receptor with high affinity and activates the Janus kinases Jak1 and Jak2, leading to engagement of Jak-Stat pathways and transcriptional activation of IFN␥-regulated genes (13-16). Activation of the Jak-Stat pathway is critical for the IFN␥ transcriptional control of IFN-stimulated genes (ISGs) 3 and, subsequently, for the generation of IFN␥-induced biological responses (13-16). Beyond the classical JakStat pathways, several other signaling pathways have been shown to be activated by the type II IFN receptor, and their function appears to be critical for IFN␥ responses. These include PKC (17), MAP kinase (18,19), and Mnk kinase cascades (20). There is evidence that the AKT/mTOR pathway is engaged in IFN␥ signaling, controlling the initiation of mRNA translation for ISGs (21,22). However, the precise contribution of different mTOR complexes in this process and the sequence of events leading to ISG mRNA translation remain to be determined.The mTOR kinase forms the catalytic core of two known complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (24 -35). mTORC1 is a protein complex consisting of mTOR, mammalian lethal with Sec 13 protein 8/G-protein ␤-protein subunit like (mLST8/G␤L), rapamycin-sensitive companion of mTOR (Raptor), Akt/PKB substrate 40 kDa (Pras40), and DEP domain-containing mTOR-interacting protein (Deptor) (24, 25). mTORC1 is known as a key regulator of pathways involved in the initiation of mRNA translation and is inhibited by allosteric inhibitors such as rapamycin, everolimus, temsirolimus, and other rapalogs (24,25). mTORC2 is comprised of mTOR, mLST8, rapamycin-insensitive companion of mTOR (Rictor), mammalian stress-activated protein kinase interacting protein 1 (Sin1), protein observed with rictor 1/2 (protor 1/2), and deptor (26 -32). Although the two mTOR complexes have different...

mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fluorescently labeled RNAs in decapping assays, RNA decay assays, and RNA visualization in cells. Finally, we also demonstrated that mRNAs labeled by the reported method are translationally active. We envisage that the novel analogs will provide an alternative to radiolabeling of mRNA caps for in vitro studies and open possibilities for new applications related to the study of mRNA fates in vivo.

Targeting cap-dependent translation initiation is one of experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5’, 5’-triphosphates cap analogs are potent antagonists of eukaryotic...