AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling

Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors

Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366–375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of β-galactosidase (β-Gal) in several mammalian cell lines, and expression remained high (∼150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by β-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-β-Gal antibody response after intramuscular inoculation of the β-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo

Monoallelic but not biallelic loss of Dicer1 promotes tumorigenesis in vivo

Human tumors are characterized by widespread reduction in microRNA (miRNA) expression, although it is unclear how such changes come about and whether they have an etiological role in the disease. Importantly, miRNA knockdown has been shown to enhance the tumorigenic potential of human lung adenocarcinoma cells. A defect in miRNA processing is one possible mechanism for global downregulation. To explore this possibility in more detail in vivo, we have manipulated Dicer1 gene dosage in a mouse model of retinoblastoma. We show that although monoallelic loss of Dicer1 does not affect normal retinal development, it dramatically accelerates tumor formation on a retinoblastoma-sensitized background. Importantly, these tumors retain one wild-type Dicer1 allele and exhibit only a partial decrease in miRNA processing. Accordingly, in silico analysis of human cancer genome data reveals frequent hemizygous, but not homozygous, deletions of DICER1. Strikingly, complete loss of Dicer1 function in mice did not accelerate retinoblastoma formation. miRNA profiling of these tumors identified members of the let-7 and miR-34 families as candidate tumor suppressors in retinoblastoma. We conclude that Dicer1 functions as a haploinsufficient tumor suppressor. This finding has implications for cancer etiology and cancer therapy