Tominori Kimura

Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes

During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1β (IL‐1β) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3′‐untranslated region (3′UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5‐kilobase region 3′‐flanking to iNOS gene exon 27 was involved in IL‐1β induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3′UTR. These results together with the data in a yeast RNA‐hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU‐rich element‐binding protein HuR, a human homolog of embryonic lethal‐abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA‐binding proteins may mutually interact. Conclusion: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA‐RNA interaction may be a new therapeutic target for NO‐mediating inflammatory diseases. (HEPATOLOGY 2008.)

Rev-dependent association of the intron-containing HIV-1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin-B.

A hallmark of HIV‐1 gene expression is that unspliced genomic RNA, which also acts as mRNA for the expression of Gag/Pol, is exported to the cytoplasm. Rev directs this transport through the nuclear export signal (NES).

CRM1-dependent, but not ARE-mediated, nuclear export of IFN-α1 mRNA

While the bulk of cellular mRNA is known to be exported by the TAP pathway, export of specific subsets of cellular mRNAs may rely on chromosome region maintenance 1 (CRM1). One line of evidence supporting this hypothesis comes from the study of mRNAs of certain early response genes (ERGs) containing the adenylate uridylate-rich element (ARE) in their 3′ untranslated regions (3′ UTRs). It was reported that HuR-mediated nuclear export of these mRNAs was CRM1-dependent under certain stress conditions. To further examine potential CRM1 pathways for other cellular mRNAs under stress conditions, the nuclear export of human interferon-α1 (IFN-α1) mRNA, an ERG mRNA induced upon viral infection, was studied. Overproduction of human immunodeficiency virus type 1 Rev protein reduced the expression level of the co-transfected IFN-α1 gene. This inhibitory effect, resulting from nuclear retention of IFN-α1 mRNA, was reversed when rev had a point mutation that made its nuclear export signal unable to associate with CRM1. Leptomycin B sensitivity experiments revealed that the cytoplasmic expression of IFN-α1 mRNA was arrested upon inhibition of CRM1. This finding was further supported by overexpression of ΔCAN, a defective form of the nucleoporin Nup214/CAN that inhibits CRM1 in a dominant-negative manner, which resulted in the effective inhibition of IFN-α1 gene expression. Subsequent RNA fluorescence in situ hybridisation and immunocytochemistry demonstrated that the IFN-α1 mRNA was colocalised with CRM1, but not with TAP, in the nucleus. These results therefore imply that the nuclear export of IFN-α1 mRNA is mediated by CRM1. However, truncation of the 3′ UTR did not negatively affect the nuclear export of IFN-α1 mRNA that lacked the ARE, unexpectedly indicating that this CRM1-dependent mRNA export may not be mediated via the ARE.

Interleukin‐1β induces tumor necrosis factor‐α secretion from rat hepatocytes

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF‐α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS).

Stabilization of human interferon-α1 mRNA by its antisense RNA.

Antisense transcription is a widespread phenomenon in the mammalian genome and is believed to play a role in regulating gene expression. However, the exact functional significance of antisense transcription is largely unknown. Here, we show that natural antisense (AS) RNA is an important modulator of interferon-α1 (IFN-α1) mRNA levels. A ~4-kb, spliced IFN-α1 AS RNA targets a single-stranded region within a conserved secondary structure element of the IFN-α1 mRNA, an element which was previously reported to function as the nuclear export element. Following infection of human Namalwa lymphocytes with Sendai virus or infection of guinea pig 104C1 fetal fibroblasts with influenza virus A/PR/8/34, expression of IFN-α1 AS RNA becomes elevated. This elevated expression results in increased IFN-α1 mRNA stability because of the cytoplasmic (but not nuclear) interaction of the AS RNA with the mRNA at the single-stranded region. This results in increased IFN-α protein production. The silencing of IFN-α1 AS RNA by sense oligonucleotides or over-expression of antisense oligoribonucleotides, which were both designed from the target region, confirmed the critical role of the AS RNA in the post-transcriptional regulation of IFN-α1 mRNA levels. This AS RNA stabilization effect is caused by the prevention of the microRNA (miRNA)-induced destabilization of IFN-α1 mRNA due to masking of the miR-1270 binding site. This discovery not only reveals a regulatory pathway for controlling IFN-α1 gene expression during the host innate immune response against virus infection but also suggests a reason for the large number of overlapping complementary transcripts with previously unknown function.

Nuclear and cytoplasmic effects of human CRM1 on HIV-1 production in rat cells

The human immunodeficiency virus type 1 (HIV‐1) regulatory protein, Rev, mediates the nuclear export of unspliced gag and singly spliced env mRNAs by bridging viral RNA and the export receptor, CRM1. Recently, rat CRM1 was found to be less efficient than human CRM1 in supporting Rev function in rats. In this study, to understand the role of CRM1 in HIV propagation, the mechanism underlying the function of human and rat CRM1 in HIV‐1 replication was investigated in rat cells. The production of viral particles, represented by the p24 Gag protein, was greatly enhanced by hCRM1 expression in rat cells; however, this effect was not simply because of the enhanced export of gag mRNA. The translation initiation rate of gag mRNA was not increased, nor was the Gag protein stabilized in the presence of hCRM1. However, the processing of the p55 Gag precursor and the release of viral particles were facilitated. These results indicated that hCRM1 exports gag mRNA to the cytoplasm, not only more efficiently than rCRM1 but also correctly, leading to efficient processing of Gag proteins and particle formation.

Bioisostere of valtrate, anti-HIV principle by inhibition for nuclear export of Rev

Rational design by the MO calculation disclosed 5,6-dihydrovaltrate (2) as the bioisostere of valtrate (1), the Rev-export inhibitor with anti-HIV activity. The synthesis of 2 was accomplished by ingenious use of asymmetric Diels-Alder reaction and stereoselective epoxidation associated with the adjacent hydroxyl group. Because of similar biological potency to 1, the analog 2 should be recognized as a promising scaffold for new anti-HIV agents with an unprecedented mechanism of action, inhibition for nuclear export of Rev protein, in the conventional remedy.

Novel cis-active structures in the coding region mediate CRM1-dependent nuclear export of IFN-α 1 mRNA.

We recently reported the chromosome region maintenance 1 (CRM1)-dependent nuclear export of intron-less human interferon-α1 (IFN-α1) mRNA, which encodes a main effecter of host innate immunity. We show that the coding region of IFN-α1 mRNA forms novel secondary structures that are responsible for the CRM1-dependent export of the transcript. Deletion-mutagenesis, in vivo export assays, and computer analyses of the folding potentials of export-competent fragments revealed the presence of a domain, termed the conserved secondary structure (CSS), comprising two adjacent putative stable stem-loop structures (nt 208–452). Internal deletion-mutagenesis and constitutive export assays of each stem-loop structure demonstrated that subregions 308–322 and 352–434 act as a core element by conferring the export function on the CSS. Leptomycin B (LMB) inhibition of the CRM1 pathway decreased the export of core element RNA, implying that the principal site of CRM1 action for exporting IFN-α1 mRNA resides within the core element. An RNPS1 (RNA-binding protein S1, serine-rich domain) cDNA was isolated by yeast three-hybrid screening, using bait containing two CSS regions. We showed that RNPS1 might recognize IFN-α1 mRNP that includes CRM1. The data demonstrate that interaction between RNA structures in the coding region and CRM1 affects the nucleocytoplasmic translocation of IFN-α1 mRNA.

Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase

Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of ΔCAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.