ABSTRACT
We report here that the alternatively spliced nuclear factors associated with double-stranded RNA, NFAR-1 (90 kDa) and -2 (110 kDa), are involved in retaining cellular transcripts in intranuclear foci and can regulate the export of mRNA to the cytoplasm. Furthermore, the NFAR proteins were found to remain associated with exported ribonucleoprotein complexes. Loss of NFAR function, which was embryonic-lethal, caused an increase in protein synthesis rates, an effect augmented by the presence of the mRNA export factors TAP, p15, or Rae1. Significantly, NFAR depletion in normal murine fibroblasts rendered these cells dramatically susceptible to vesicular stomatitis virus replication. Collectively, our data demonstrate that the NFARs exert influence on mRNA trafficking and the modulation of translation rates and may constitute an innate immune translational surveillance mechanism important in host defense countermeasures against virus infection.
Subject(s)
Nuclear Factor 90 Proteins/metabolism , Protein Biosynthesis/genetics , Animals , Cells, Cultured , Gene Deletion , Humans , Mice , Nuclear Factor 90 Proteins/genetics , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , RNA Interference , RNA, Messenger/genetics , RNA, Small Interfering/geneticsABSTRACT
The connexin43 (cx43) gene was originally described as consisting of two exons, one coding for most of the 5'-untranslated region (5'-UTR), and the other for the protein sequence and 3'-UTR. We now report that in mouse four additional exons are expressed, all coding for novel 5'-UTRs. Altogether, we found nine different cx43 mRNA species (GenBank accession numbers NM010288, and AY427554 through AY427561) generated by differential promoter usage and alternative splicing mechanisms. The relative abundance of these different mRNAs varied with the tissue source. In addition, the different transcripts showed varying translational efficiencies in several cell lines, indicating the presence of cis-RNA elements that regulate cx43 translation. We propose that it is the promoter driving the expression of the cx43 gene that determines exon choice in the downstream splicing events in a cell-type-dependent fashion. This in turn will affect the translation efficiency of the transcript orchestrating the events that lead to the final expression profile of cx43. Since a similar organization of the cx43 gene was also observed in rat it is likely that the complex regulation of cx43 expression involving transcription, splicing and translation mechanisms is a common trait conserved during evolution.
Subject(s)
Alternative Splicing , Connexin 43/genetics , Promoter Regions, Genetic , Protein Biosynthesis , 5' Untranslated Regions , Animals , Base Sequence , Cell Line , Codon , Connexin 43/metabolism , Cricetinae , Exons , Gene Expression Regulation , Humans , Mice , Molecular Sequence Data , RNA, Messenger/metabolism , Rats , Regulatory Sequences, Ribonucleic Acid , Transcription Initiation Site , Transcription, GeneticABSTRACT
This article describes the structural and functional characterization of Ini (AF495522), a novel highly conserved zinc-finger protein that had been identified by screening an estrogen-induced rat myometrial expression library. Ini localizes to the nucleus of HeLa cells and binds to the proximal connexin43 (cx43) promoter, as demonstrated by EMSA. In addition, transient transfection experiments performed with estrogen receptor alpha (ERalpha) cDNA show that overexpression of Ini enhances, in a dose-dependent fashion, the up-regulation of the cx43 gene by estrogen. On binding to the cx43 promoter, Ini stimulates the transcriptional activating function (AF)-1, but not the AF-2, of the ERalpha. This makes Ini one of the few known coactivators specific for AF-1. Because estrogen up-regulates Ini mRNA in the myometrium, it is likely that Ini's physiological role in this tissue is to modulate the response of the cx43 gene to estrogen. Transfection studies with an Ini antisense construct seem to indicate that Ini plays an additional role in the cellular response to estrogen affecting both AF-1 and AF-2 activities of the ERalpha. This broader effect may be associated with cell cycle progression that in yeast has been shown to require Ini.
Subject(s)
Connexin 43/genetics , Estrogens/pharmacology , Nuclear Proteins/genetics , Zinc Fingers/genetics , Animals , COS Cells , Carcinoma, Hepatocellular , Cell Nucleus/metabolism , Conserved Sequence , Estrogen Receptor alpha , Evolution, Molecular , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , HeLa Cells , Humans , Liver Neoplasms , Molecular Sequence Data , Nuclear Proteins/metabolism , Promoter Regions, Genetic/physiology , Protein Structure, Tertiary , Rats , Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Sequence Homology, Amino Acid , Up-Regulation/drug effects , Uterus/physiologyABSTRACT
Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3'-azido-3'-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that pi-pi interactions between RT and an aromatic structure may be facilitated by these mutations.
