Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A) ⍣ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.
In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.
Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single ∼35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.
Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.
Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3' overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt-like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3' overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.
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