Exportin-5 mediates nuclear export of minihelix-containing RNAs

The adenovirus VA1 RNA (VA1), a 160-nucleotide (nt)-long RNA transcribed by RNA polymerase III, is efficiently exported from the nucleus to the cytoplasm of infected cells, where it antagonizes the interferon-induced antiviral defense system. We recently reported that nuclear export of VA1 is mediated by a cis-acting RNA export motif, called minihelix, that comprises a double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt protruding 3' end. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin) that remained to be determined. Here we show using microinjection in Xenopus laevis oocytes that VA1 is transported to the cytoplasm by exportin-5, a nuclear transport factor for double-stranded RNA binding proteins. Gel retardation assays revealed that exportin-5 directly interacts with VA1 RNA in a RanGTP-dependent manner. More generally, in vivo and in vitro competition experiments using various VA1-derived, but also artificial and cellular, RNAs lead to the conclusion that exportin-5 preferentially recognizes and transports minihelix motif-containing RNAs.     

Dynamic mechanisms for pre-miRNA binding and export by Exportin-5

The biogenesis and function of mature microRNAs (miRNAs) is dependent on the nuclear export of miRNA precursors (pre-miRNA) by Exportin-5 (Exp5). To characterize the molecular mechanisms of how pre-miRNA is recognized and transported by Exp5, we have performed 21 molecular dynamic (MD) simulations of RNA-bound Exp5 (Exp5-RanGTP-premiRNA, Exp5-RanGDP-premiRNA, Exp5-premiRNA), RNA-unbound Exp5 (Exp5-RanGTP, Exp5-RanGDP, apo-Exp5), and pre-miRNA. Our simulations with standard MD, steered molecular dynamics (SMD), and energy analysis have shown that (1) Free Exp5 undergoes extensive opening motion, and in this way facilitates the RanGTP binding. (2) RanGTP efficiently regulates the association/dissociation of pre-miRNA to its complex by inducing conformational changes in the HEAT-repeat helix stacking of Exp5. (3) The GTP hydrolysis prevents Ran from rebinding to Exp5 by regulating the hydrophobic interfaces and salt bridges between Ran and Exp5. (4) The transition from the A'-form to the A-form of the pre-miRNA modulates the structural complementarities between the protein and the pre-miRNA, thus promoting efficient assembly of the complex. (5) The base-flipping process (from the closed to the fully flipped state) of the 2-nt 3' overhang is a prerequisite for the pre-miRNA recognition by Exp5, which occurs in a sequence-independent manner as evidenced by the fact that different 2-nt 3' overhangs bind to Exp5 in essentially the same way. And finally, a plausible mechanism of the pre-miRNA export cycle has been proposed explaining how the protein-protein and protein-RNA interactions are coordinated in physiological conditions.     

Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins.

We have identified a novel human karyopherin (Kap) beta family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.     

Interaction of the eukaryotic elongation factor 1A with newly synthesized polypeptides

eEF1A, the eukaryotic homologue of bacterial elongation factor Tu, is a well characterized translation elongation factor responsible for delivering aminoacyl-tRNAs to the A-site at the ribosome. Here we show for the first time that eEF1A also associates with the nascent chain distal to the peptidyltransferase center. This is demonstrated for a variety of nascent chains of different lengths and sequences. Interestingly, unlike other ribosome-associated factors, eEF1A also interacts with polypeptides after their release from the ribosome. We demonstrate that eEF1A does not bind to correctly folded full-length proteins but interacts specifically with proteins that are unable to fold correctly in a cytosolic environment. This association was demonstrated both by photo-cross-linking and by a functional refolding assay.     

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.     

CRM1-mediated nuclear export: to the pore and beyond

CRM1 (chromosome region maintenance 1; also referred to as exportin1 or Xpo1) is a member of the importin beta superfamily of nuclear transport receptors, recognizing proteins bearing a leucine-rich nuclear export sequence. CRM1 is the major receptor for the export of proteins out of the nucleus and is also required for transport of many RNAs. Besides its established role in nuclear export, CRM1 is also implicated in various steps during mitosis, widening its functional spectrum within the cell.     

Putative reaction intermediates in Crm1-mediated nuclear protein export

We discovered several novel interactions between proteins involved in Crm1-mediated nuclear export of the nuclear export signal containing human immunodeficiency virus type 1 protein Rev. First, a Rev/Crm1/RanGTP complex (where Ran is Ras-related nuclear protein) reacts with some nucleoporins (Nup42 and Nup159) but not others (NSP1, Nup116, and Nup1), forming a Nup/Crm1/RanGTP complex and concomitantly releasing Rev. Second, RanBP1 (or homologous proteins) can displace Nup and form a ternary RanBP1/RanGTP/Crm1 complex that can be disassembled by RanGAP via GTP hydrolysis. Third, and most surprisingly, RanBP1/RanGTP/Crm1 can be disassembled without GTP hydrolysis by the nucleotide exchange factor RanGEF. Recycling of a Ran/RanGEF complex by GTP and Mg2+ is stimulated by both Crm1 and Rev, allowing reformation of a Rev/Crm1/RanGTP complex. Based on these reactions we propose a model for Crm1-mediated export.     

A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.     

A non-canonical function of eukaryotic elongation factor 1A1: Regulation of interleukin-6 expression

Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3^S727 phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.     

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.     

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.     

A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.

Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.     

Stm1p alters the ribosome association of eukaryotic elongation factor 3 and affects translation elongation

Stm1p is a Saccharomyces cerevisiae protein that is primarily associated with cytosolic 80S ribosomes and polysomes. Several lines of evidence suggest that Stm1p plays a role in translation under nutrient stress conditions, although its mechanism of action is not yet known. In this study, we show that yeast lacking Stm1p (stm1Δ) are hypersensitive to the translation inhibitor anisomycin, which affects the peptidyl transferase reaction in translation elongation, but show little hypersensitivity to other translation inhibitors such as paromomycin and hygromycin B, which affect translation fidelity. Ribosomes isolated from stm1Δ yeast have intrinsically elevated levels of eukaryotic elongation factor 3 (eEF3) associated with them. Overexpression of eEF3 in cells lacking Stm1p results in a growth defect phenotype and increased anisomycin sensitivity. In addition, ribosomes with increased levels of Stm1p exhibit decreased association with eEF3. Taken together, our data indicate that Stm1p plays a complementary role to eEF3 in translation.     

Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells

We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.