Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Structure-based phylogeny of class IIa tRNA synthetases in relation to an unusual biochemistry.

The available three-dimensional information for class II aminoacyl-tRNA synthetases has been used to generate sequence alignments that strictly adhere to the structural equivalencies between members of subclass IIa of these enzymes. The resulting alignments were used to study their phylogenetic relationships. In particular, the entire set of available sequences of prolyl-tRNA synthetases was analyzed in this way. In contrast to recent reports, we conclude that the evolutionary pattern of prolyl-tRNA synthetases does not obviously conform to the canonical phylogenetic distribution. The pattern found for these enzymes may be related to their biochemical characteristics. Our results indicate a potential relationship between the evolutionary pattern of prolyl-tRNA synthetases and the emergence of two enzymatically distinct forms of these proteins.

Aminoacyl-tRNA synthetases: potential markers of genetic code development.

Aminoacylation of tRNAs, catalyzed by 20 aminoacyl-tRNA synthetases, is responsible for establishing the genetic code. The enzymes are divided into two classes on the basis of the architectures of their active sites. Members of the two classes also differ in that they bind opposite sides of the tRNA acceptor stem. Importantly, specific pairs of synthetases--one from each class--can be docked simultaneously onto the acceptor stem. This article relates these specific pairings to the organization of the table of codons that defines the universal genetic code.

Origin of mitochondria in relation to evolutionary history of eukaryotic alanyl-tRNA synthetase.

The origin of the eukaryotic cell remains an unsolved question. Numerous experimental and phylogenetic observations support the symbiotic origin of the modern eukaryotic cell, with its nucleus and (typically) mitochondria. Incorporation of mitochondria has been proposed to precede development of the nucleus, but it is still unclear whether mitochondria were initially part of basal eukaryotes. Data on alanyl-tRNA synthetase from an early eukaryote and other sources are presented and analyzed here. These data are consistent with the notion that mitochondrial genesis did not significantly precede nucleus formation. Moreover, the data raise the possibility that diplomonads are primary amitochondriates that radiated from the eukaryotic lineage before mitochondria became fully integrated as a cellular organelle.

An ancestral MADS-box gene duplication occurred before the divergence of plants and animals.

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle.

Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo.

Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains.

RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.

Genetic code origins: tRNAs older than their synthetases?

We present a phylogenetic analysis to determine whether a given tRNA molecule was established in evolution before its cognate aminoacyl-tRNA synthetase. The earlier appearance of tRNA versus their metabolically related enzymes is a prediction of the RNA world theory, but the available synthetase and tRNA sequences previously had not allowed a formal comparison of their relative time of appearance. Using data recently obtained from the emerging genome projects, our analysis points to the extant forms of lysyl-tRNA synthetase being preceded in evolution by the establishment of the identity of lysine tRNA.

Nup84, a novel nucleoporin that is associated with CAN/Nup214 on the cytoplasmic face of the nuclear pore complex.

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled-coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled-coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH2-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.

Species-specific tRNA recognition in relation to tRNA synthetase contact residues.

In spite of variations in the sequences of tRNAs, the genetic code (anticodon trinucleotides) is conserved in evolution. However, non-anticodon nucleotides which are species specific are known to prevent a given tRNA from functioning in all organisms. Conversely, species-specific tRNA contact residues in synthetases should also prevent cross-species acylation in a predictable way. To address this question, we investigated the relatively small tyrosine tRNA synthetase where contacts of Escherichia coli tRNA(Tyr) with the alpha2 dimeric protein have been localized by others to four specific sequence clusters on the three-dimensional structure of the Bacillus stearothermophilus enzyme. We used specific functional tests with a previously not-sequenced and not-characterized Mycobacterium tuberculosis enzyme and showed that it demonstrates species-specific aminoacylation in vivo and in vitro. The specificity observed fits exactly with the presence of the clusters characteristic of those established as important for recognition of E. coli tRNA. Conversely, we noted that a recent analysis of the tyrosine enzyme from the eukaryote pathogen Pneumocystis carinii showed just the opposite species specificity of tRNA recognition. According to our alignments, the sequences of the clusters diverge substantially from those seen with the M. tuberculosis, B. stearothermophilus and other enzymes. Thus, the presence or absence of species-specific residues in tRNA synthetases correlates in both directions with cross-species aminoacylation phenotypes, without reference to the associated tRNA sequences. We suggest that this kind of analysis can identify those synthetase-tRNA covariations which are needed to preserve the genetic code. These co-variations might be exploited to develop novel antibiotics against pathogens such as M. tuberculosis and P. carinii.

Reconstruction of quaternary structures of class II tRNA synthetases by rational mutagenensis of a conserved domain.

Class II tRNA synthetases have long been known to have quaternary structures of alpha, alpha2, alpha2beta2, and alpha4, depending on the amino acid specificity and the organism from which the synthetase was isolated. Even the quaternary structures of enzymes for the same amino acid show variations in evolution. The basis for these variations has not been understood. We report here that sequence manipulations of a structural motif (motif 1) characteristic of all class II tRNA synthetases can generate most of the evolutionary diversity of quaternary forms of class II synthetases. Thus, the principles elucidated here for quaternary structure assembly may be general.

Developmentally regulated alternative splicing of mRNAs encoding N-terminal tau variants in the rat hippocampus: structural and functional implications.

Tau protein variants are axonal microtubule-associated phosphoproteins whose expression correlates with developmentally regulated neurite outgrowth. A single gene encodes multiple tau transcripts via complex alternative splicing. We studied the expression of the mRNAs encoding N-terminal variants of tau, and we showed distinct alternative splicing of exons 2 and 3 in nervous tissues of the adult rat, including the inner ear, hippocampus, cortex, striatum, brainstem, cerebellum, olfactory bulb and retina. Using the reverse transcriptase-coupled polymerase chain reaction and in situ hybridization, we then focused our developmental study on hippocampal neurons, both in vivo and in vitro, to address the developmental and spatial expression of the alternatively spliced mRNAs encoding N-terminal variants of tau. Tau mRNAs devoid of exons 2 and 3 were present throughout development, although their levels decreased in adults. Those containing exon 2 but not exon 3 were already present in the hippocampus of newborn rats and their levels increased during the first postnatal week, mainly in the pyramidal cell layer. Tau RNAs containing exons 2 and 3 appeared at the end of this period in the pyramidal cell layer and in the dentate granule cells. Exon 2-containing mRNAs seemed to be associated with cells undergoing axonal sprouting, while exon 3-containing RNAs were expressed in mature neurons that had established their connections. The timing and pattern of tau alternative splicing were maintained in cultured hippocampal neurons, suggesting that splicing processes are independent of the organized connectivity and of the environmental cues provided in vivo. Secondary structure predictions of tau variants revealed that the insertion of the exon 3-encoded domain substantially modifies the secondary structure of the N-terminal region of tau. This N-terminal heterogeneity may confer distinct regulatory roles on the tau variants during ontogeny and may contribute to plasticity in the adult rat brain.