Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

Intron retention is one of the least studied forms of alternative splicing. Through the use of retrovirus and other model systems, it was established many years ago that mRNAs with retained introns are subject to restriction both at the level of nucleocytoplasmic export and cytoplasmic expression. It was also demonstrated that specific cis-acting elements in the mRNA could serve to bypass these restrictions. Here we show that one of these elements, the constitutive transport element (CTE), first identified in the retrovirus MPMV and subsequently in the human NXF1 gene, is a highly conserved element. Using GERP analysis, CTEs with strong primary sequence homology, predicted to display identical secondary structure, were identified in NXF genes from >30 mammalian species. CTEs were also identified in the predicted NXF1 genes of zebrafish and coelacanths. The CTE from the zebrafish NXF1 was shown to function efficiently to achieve expression of mRNA with a retained intron in human cells in conjunction with zebrafish Nxf1 and cofactor Nxt proteins. This demonstrates that all essential functional components for expression of mRNA with retained introns have been conserved from fish to man.     

Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway

The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27-mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP-mediated export pathway. This represents a novel mechanism for export of viral mRNAs.     

Following the leader: bacterial protein export through the Sec pathway.

Significant strides have been made during the past 20 years in our understanding of protein secretion across the bacterial inner membrane. Specialized chaperones select secretory polypeptide chains and usher them to a membrane-embedded preprotein translocase. This unique molecular machine envelops the polymeric substrate and migrates along its length in defined, energy-dependent steps. Consequently, preproteins are gradually pumped into the periplasm where they acquire their native, folded conformation.     

Export of adenoviral late mRNA from the nucleus requires the Nxf1/Tap export receptor

One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infected-cell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.     

The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A).

The influenza virus NS1 protein inhibits the nuclear export of a spliced viral mRNA, NS2 mRNA (F. V. Alonso-Caplen, M. E. Nemeroff, Y. Qiu, and R. M. Krug, Genes Dev. 6:255-267, 1992). To identify the sequence in NS2 mRNA that is recognized by the NS1 protein, we developed a gel shift assay for the formation of specific RNA-protein complexes. With this assay, it was established that the NS1 protein binds to the poly(A) sequence at the 3' end of NS2 mRNA and of other mRNAs. In addition, the NS1 protein was shown to bind to poly(A) itself. This specificity was also observed in vivo. The NS1 protein inhibited the nuclear export of every poly(A)-containing mRNA that was tested. In contrast, the NS1 protein failed to inhibit the nuclear export of an mRNA whose 3' end was generated by cleavage without subsequent addition of poly(A). Addition of poly(A) to this mRNA enabled the NS1 protein to inhibit mRNA export. The implications of these results for the role of the NS1 protein during virus infection are discussed.     

The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gbetagamma

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1alpha and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1alpha-induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1alpha receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gbetagamma-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gbetagamma function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gbetagamma in pull downs. Interaction with Gbetagamma did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1alpha activation of phospholipase C, indicating that PLIC-1's inhibition of Gbetagamma did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gbetagamma; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.     

The J domain of Tpr2 regulates its interaction with the proapoptotic and cell-cycle checkpoint protein, Rad9

Human Rad9 is a key cell-cycle checkpoint protein that is postulated to function in the early phase of cell-cycle checkpoint control through complex formation with Rad1 and Hus1. Rad9 is also thought to be involved in controlling apoptosis through its interaction with Bcl-2. To explore the biochemical functions of Rad9 in these cellular control mechanisms, we performed two-hybrid screening and identified Tetratricopeptide repeat protein 2 (Tpr2) as a novel Rad9-binding protein. We found that Tpr2 binds not only to Rad9, but also to Rad1 and Hus1, through its N-terminal tetratricopeptide repeat region, as assessed by in vivo and in vitro binding assays. However, the in vivo and in vitro interactions of Tpr2 with Rad9 were greatly enhanced by the deletion of its C-terminal J domain or by a point mutation in the conserved HPD motif in the J domain, though the binding of Tpr2 to Rad1 and Hus1 was not influenced by these J-domain mutations. We further found: (1) Rad9 transiently dissociates from Tpr2 following heat-shock or UV treatments, but the mutation of the J domain abrogates this transient dissociation of the Tpr2/Rad9 complex; and (2) the J domain of Tpr2 modulates the cellular localization of both Tpr2 itself and Rad9. These results indicate that the J domain of Tpr2 plays a critical role in the regulation of both physical and functional interactions between Tpr2 and Rad9.     

The inherited blindness protein AIPL1 regulates the ubiquitin-like FAT10 pathway

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

ICP27 interacts with the RNA export factor Aly/REF to direct herpes simplex virus type 1 intronless mRNAs to the TAP export pathway

Herpes simplex virus type 1 (HSV-1) protein ICP27 facilitates the export of viral intronless mRNAs. ICP27 shuttles between the nucleus and cytoplasm, which has been shown to require a leucine-rich nuclear export sequence (NES). ICP27 export was reported to be sensitive to the CRM1 inhibitor leptomycin B (LMB) in HSV-1-infected cells but not in Xenopus oocytes, where ICP27 interacts with the export factor Aly/REF to access the TAP export pathway. Here, we show that ICP27 interacts with Aly/REF in HSV-1-infected mammalian cells and that Aly/REF stimulates export of viral intronless RNAs but does not cross-link to these RNAs. During infection, Aly/REF was no longer associated with splicing factor SC35 but moved into structures that colocalized with ICP27, suggesting that ICP27 recruits Aly/REF from spliceosomes to viral intronless RNAs. Further, ICP27 was found to interact in vivo with TAP but not with CRM1. In vitro export assays showed that ICP27 export was not sensitive to LMB but was blocked by a dominant-negative TAP deletion mutant lacking the nucleoporin interaction domain. These data suggest that ICP27 uses the TAP pathway to export viral RNAs. Interestingly, the leucine-rich N-terminal sequence was required for efficient export, even though ICP27 export was LMB insensitive. Thus, this region is required for efficient ICP27 export but does not function as a CRM1-dependent NES.     

The sphingolipid pathway regulates Pkc1 through the formation of diacylglycerol in Cryptococcus neoformans

The sphingolipid biosynthetic pathway generates bioactive molecules crucial to the regulation of mammalian and fungal physiological and pathobiological processes. In previous studies (Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R., Hannun, Y. A., and Del Poeta, M. (2001) Genes Dev. 15, 201-212), we demonstrated that an enzyme of the fungal sphingolipid pathway, Ipc1 (inositol-phosphorylceramide synthase-1), regulates melanin, a pigment required for the pathogenic fungus Cryptococcus neoformans to cause disease. In this study, we investigated the mechanism by which Ipc1 regulates melanin production. Because Ipc1 also catalyzes the production of diacylglycerol (DAG), a physiological activator of the classical and novel isoforms of mammalian protein kinase C (PKC), and because it has been suggested that PKC is required for melanogenesis in mammalian cells, we investigated whether Ipc1 regulates melanin in C. neoformans through the production of DAG and the subsequent activation of Pkc1, the fungal homolog of mammalian PKC. The results show that modulation of Ipc1 regulates the levels of DAG in C. neoformans cells. Next, we demonstrated that C. neoformans Pkc1 is a DAG-activated serine/threonine kinase and that the C1 domain of Pkc1 is necessary for this activation. Finally, through both pharmacological and genetic approaches, we found that inhibition of Pkc1 abolishes melanin formation in C. neoformans. This study identifies a novel signaling pathway in which C. neoformans Ipc1 plays a key role in the activation of Pkc1 through the formation of DAG. Importantly, this pathway is essential for melanin production with implications for the pathogenicity of C. neoformans.     

Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway

The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFkappaB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFkappaB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis.     

The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses

The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. Here, we show that FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses could result in synaptic dysfunction phenotype of Fragile X patients.     

Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.     

GL3 encodes a bHLH protein that regulates trichome development in arabidopsis through interaction with GL1 and TTG1

Arabidopsis trichome development and differentiation is a well-studied model for plant cell-fate determination and morphogenesis. Mutations in TRANSPARENT TESTA GLABRA1 (TTG1) result in several pleiotropic defects including an almost complete lack of trichomes. The complex phenotype caused by ttg1 mutations is suppressed by ectopic expression of the maize anthocyanin regulator R. Here it is demonstrated that the Arabidopsis trichome development locus GLABRA3 (GL3) encodes an R homolog. GL3 and GLABRA1 (GL1) interact when overexpressed together in plants. Yeast two-hybrid assays indicate that GL3 participates in physical interactions with GL1, TTG1, and itself, but that GL1 and TTG1 do not interact. These data suggest a reiterated combinatorial model for the differential regulation of such diverse developmental pathways as trichome cell-fate determination, root hair spacing, and anthocyanin secondary metabolism.     

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.     

The unfolded protein response regulates glutamate receptor export from the endoplasmic reticulum

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate the majority of excitatory signaling in the CNS, and the functional properties and subcellular fate of these receptors depend on receptor subunit composition. Subunit assembly is thought to occur in the endoplasmic reticulum (ER), although we are just beginning to understand the underlying mechanism. Here we examine the trafficking of Caenorhabditis elegans glutamate receptors through the ER. Our data indicate that neurons require signaling by the unfolded protein response (UPR) to move GLR-1, GLR-2, and GLR-5 subunits out of the ER and through the secretory pathway. In contrast, other neuronal transmembrane proteins do not require UPR signaling for ER exit. The requirement for the UPR pathway is cell type and age dependent: impairment for receptor trafficking increases as animals age and does not occur in all neurons. Expression of XBP-1, a component of the UPR pathway, is elevated in neurons during development. Our results suggest that UPR signaling is a critical step in neural function that is needed for glutamate receptor assembly and secretion.     

The cAMP signaling pathway regulates Epe1 protein levels and heterochromatin assembly

The epigenetic landscape of a cell frequently changes in response to fluctuations in nutrient levels, but the mechanistic link is not well understood. In fission yeast, the JmjC domain protein Epe1 is critical for maintaining the heterochromatin landscape. While loss of Epe1 results in heterochromatin expansion, overexpression of Epe1 leads to defective heterochromatin. Through a genetic screen, we found that mutations in genes of the cAMP signaling pathway suppress the heterochromatin defects associated with Epe1 overexpression. We further demonstrated that the activation of Pka1, the downstream effector of cAMP signaling, is required for the efficient translation of epe1⁺ mRNA to maintain Epe1 overexpression. Moreover, inactivation of the cAMP-signaling pathway, either through genetic mutations or glucose deprivation, leads to the reduction of endogenous Epe1 and corresponding heterochromatin changes. These results reveal the mechanism by which the cAMP signaling pathway regulates heterochromatin landscape in fission yeast.