Identification of 40LoVe, a Xenopus hnRNP D family protein involved in localizing a TGF-beta-related mRNA during oogenesis

Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromatography approach, we investigated localization RNP formation on the vegetal localization element (VLE) of the mRNA encoding Vg1, a Xenopus TGF-beta family member. We identified 40LoVe, an hnRNP D family protein, as a specific VLE binding protein from Xenopus oocytes. Interaction of 40LoVe with the VLE strictly correlates with the ability of the RNA to localize, and antibodies against 40LoVe inhibit vegetal localization in vivo in oocytes. Our results associate an hnRNP D protein with mRNA localization and have implications for several functions mediated by this important protein family.     

40LoVe interacts with Vg1RBP/Vera and hnRNP I in binding the Vg1-localization element

Localizing mRNAs within the cytoplasm gives cells the ability to spatially restrict protein production, a powerful means to regulate gene expression. Localized mRNA is often visible in microscopically observable particles or granules, and the association of mRNA localization with these structures is an indication that particles or granules may be essential to the localization process. Understanding how such structures form will therefore be important for understanding the function of localization RNPs (L-RNPs). We previously identified a novel component of an L-RNP from the Vg1 mRNA from Xenopus oocytes called 40LoVe. 40LoVe interaction with the Vg1-localization element (Vg1LE) was previously shown to be dependent on the VM1 and E2 sequence motifs within the Vg1LE that cross-link to hnRNP I and Vg1RBP/Vera, respectively. We report interaction of these motif-binding proteins with 40LoVe and identify a 40LoVe-Xenopus hnRNP D/AUF1 interaction. We further demonstrate that titration of VM1 and E2 motif binding activity in vivo surprisingly suggests that the motif binding proteins have differing roles during Vg1LE-dependent mRNA localization.     

Myosin Heavy Chains IIa and IId Are Functionally Distinct in the Mouse

Myosin in adult murine skeletal muscle is composed primarily of three adult fast myosin heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa– containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.     

Yeast hnRNP K-Like Genes Are Involved in Regulation of the Telomeric Position Effect and Telomere Length

Mammalian heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA- and DNA-binding protein implicated in the regulation of gene expression processes. To better understand its function, we studied two Saccharomyces cerevisiae homologues of the human hnRNP K, PBP2 and HEK2 (heterogeneous nuclear RNP K-like gene). pbp2Delta and hek2Delta mutations inhibited expression of a marker gene that was inserted near telomere but not at internal chromosomal locations. The telomere proximal to the ectopic marker gene became longer, while most of the other telomeres were not altered in the double mutant cells. We provide evidence that telomere elongation might be the primary event that causes enhanced silencing of an adjacent reporter gene. The telomere lengthening could, in part, be explained by the inhibitory effect of hek2Delta mutation on the telomeric rapid deletion pathway. Hek2p was detected in a complex with chromosome regions proximal to the affected telomere, suggesting a direct involvement of this protein in telomere maintenance. These results identify a role for hnRNP K-like genes in the structural and functional organization of telomeric chromatin in yeast.     

Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (αAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in αAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric α-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a β barrel structural motif.Many age-related neurodegenerative diseases are characterized by the accumulation of amyloid deposits derived from a variety of misfolded proteins (1). These diseases typically have both sporadic and inherited forms, and in many cases the mutations associated with the familial forms are in the gene encoding the protein that accumulates or in genes directly related to its production, processing, or accumulation (2). The genetic linkage between the mutant allele and disease is evidence of the causal relationship of amyloid accumulation to pathogenesis, and many of the mutations either destabilize the natively folded state, produce more amyloidogenic protein, or they increase its propensity to aggregate (3). Although fibrillar amyloid deposits are among the most obvious pathognomonic features of disease, their role in pathogenesis is not clear. The extent of fibrillar amyloid plaque deposition does not correlate well with Alzheimer''s disease pathogenesis, and there are a significant number of non-demented individuals that have equivalent amounts of amyloid plaques as disease patients (4). Pathological changes are observed in transgenic animals before the onset of amyloid plaque accumulation (5, 6), and it has been reported that soluble Aβ oligomers correlate better with dementia than insoluble, fibrillar deposits (7, 8), suggesting that oligomeric forms of Aβ may represent the primary toxic species. Soluble oligomers have been implicated as the primary toxic species in many degenerative diseases where the accumulation of large fibrillar deposits may be either inert, protective, or pathological by a different mechanism (for review, see Refs. 9 and 10).Aβ aggregates have been described ranging in size from dimers up to particles of one million daltons or larger (11–16). In the atomic force microscope prefibrillar oligomers (PFOs)³ appear as spherical particles of ∼3–10 nm. PFOs appear at early times of incubation and disappear as mature fibrils appear (16–18). At longer times of incubation PFOs appear to coalesce to form curvilinear beaded strings that have been called protofibrils and ring-shaped, pore-like structures referred to as annular protofibrils (APFs) (17). APFs appear to be formed from the circularization of PFO subunits. A similar spectrum of PFOs and APFs has been observed for many types of amyloids, such as α-synuclein (19), islet amyloid (20), and non-disease associated “neoamyloids” (21). Although PFOs, APFs, and fibrils have been observed for many different types of amyloidogenic proteins and peptides (22), their structures, interrelationships, and contributions to disease pathogenesis are not entirely clear.Insoluble fibrils and small soluble pieces of fibrils known as fibrillar oligomers appear to have a distinct and mutually exclusive underlying structure than PFOs because they display generic epitopes that are recognized by distinct conformation-dependent monoclonal antibodies (23, 24) and antisera (25, 26). It is not yet known whether APFs represent a unique conformation or whether they are structurally related to PFOs or fibrils. So far APFs have only been defined morphologically as pore-like structures and have been observed in preparations of PFOs and in fibril-containing preparations (27–29). Familial mutations associated with inherited forms of Parkinson and Alzheimer diseases increase the formation of APFs, suggesting that their formation is related to pathogenic activity (17, 30). Based on the close resemblance between APFs and bacterial pore-forming toxins, it has been proposed that APFs permeabilize membranes (22). Because membrane permeabilization is a common pathogenic activity of prefibrillar amyloid oligomers (31) and PFOs are a precursor to annular protofibril formation, the formation of APFs is an attractive explanation for the membrane permeabilization of oligomers because annular protofibril formation is also a common assembly state and they resemble pores morphologically.Investigating the pathological properties of Aβ APFs has been impeded by a lack of homogeneous preparations of annular structures and the lack of a facile means of distinguishing them from other aggregations states in vivo. Here we report the preparation of relatively homogeneous populations of APFs that have the same pore-like morphology previously described. We have used these preparations to examine their aggregation potential and membrane-permeabilizing properties and as an immunogen for the preparation of an antiserum that selectively recognizes APFs, compared with monomers, PFOs, and fibrils. APFs are stable and do not convert into fibrils or PFOs within months of incubation. APFs also exhibit much lower membrane-permeabilizing activity compared with the prefibrillar oligomer precursors to APF formation. Interaction with a hydrophobic-hydrophilic interface accelerates the conversion of PFOs into APFs. Incubation of PFOs with lipid vesicles results in a rapid loss of the prefibrillar oligomer specific epitope and the coordinate appearance of an annular protofibril-specific epitope. APFs display a unique conformation-dependent epitope that is distinct from PFOs and fibrils. Anti-annular protofibril antibody recognizes mature heptameric pores from α-hemolysin, suggesting that APFs may form β-barrel pore structures.     

Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.     

Neural inducing factors from Xenopus laevis eggs and embryos

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula-neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.     

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant.In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.     

The Origin DNA-Binding and Single-Stranded DNA-Binding Domains of Simian Virus 40 Large T Antigen Are Distinct

Little is known about the ability of simian virus 40 (SV40) T antigen to bind single-stranded DNA. We demonstrate here that a mutant (259-708) missing the first 258 amino acids of T antigen and its origin-binding domain bound single-stranded DNA at close to normal levels, whereas a mutant containing only the first 259 amino acids failed to bind any single-stranded DNA. The 259-708 mutant also assembled into high-molecular-weight oligomers in the presence of single-stranded DNA. Its ATPase activity was stimulated by single-stranded DNA similarly to the wild type (WT). Furthermore, WT T antigen’s ability to bind to single-stranded DNA was inhibited by the binding of two monoclonal antibodies that recognize a region after residue 362. These results show that the domain responsible for binding to single-stranded DNA is completely separate from the origin-binding domain.     

Distinct origins of adult and embryonic blood in Xenopus

Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.     

Cytological and Morphological Analyses Reveal Distinct Features of Intestinal Development during Xenopus tropicalis Metamorphosis

Background

The formation and/or maturation of adult organs in vertebrates often takes place during postembryonic development, a period around birth in mammals when thyroid hormone (T3) levels are high. The T3-dependent anuran metamorphosis serves as a model to study postembryonic development. Studies on the remodeling of the intestine during Xenopus (X.) laevis metamorphosis have shown that the development of the adult intestine involves de novo formation of adult stem cells in a process controlled by T3. On the other hand, X. tropicalis, highly related to X. laevis, offers a number of advantages for studying developmental mechanisms, especially at genome-wide level, over X. laevis, largely due to its shorter life cycle and sequenced genome. To establish X. tropicalis intestinal metamorphosis as a model for adult organogenesis, we analyzed the morphological and cytological changes in X. tropicalis intestine during metamorphosis.

Methodology/Principal Findings

We observed that in X. tropicalis, the premetamorphic intestine was made of mainly a monolayer of larval epithelial cells surrounded by little connective tissue except in the single epithelial fold, the typhlosole. During metamorphosis, the larval epithelium degenerates and adult epithelium develops to form a multi-folded structure with elaborate connective tissue and muscles. Interestingly, typhlosole, which is likely critical for adult epithelial development, is present along the entire length of the small intestine in premetamorphic tadpoles, in contrast to X. laevis, where it is present only in the anterior 1/3. T3-treatment induces intestinal remodeling, including the shortening of the intestine and the typhlosole, just like in X. laevis.

Conclusions/Significance

Our observations indicate that the intestine undergoes similar metamorphic changes in X. laevis and X. tropicalis, making it possible to use the large amount of information available on X. laevis intestinal metamorphosis and the genome sequence information and genetic advantages of X. tropicalis to dissect the pathways governing adult intestinal development.     

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation.Cap-dependent translation in eukaryotes begins with recognition of the 7-methylguanosine cap at the 5′ end of an mRNA by the translation initiation factor eIF4E, which forms the eIF4F complex with the scaffolding protein eIF4G. The binding of the RNA helicase eIF4A along with eIF4B promotes unwinding of mRNA secondary structure (Aitken and Lorsch, 2012). The eIF4F complex then serves to circularize mRNA by interaction of eIF4G with poly(A) binding protein and recruit the preinitiation complex through binding of eIF4G to eIF3 and eIF5, ultimately leading to the assembly of the 80S ribosome (Aitken and Lorsch, 2012). eIF4E is an attractive target for global regulation of translational activity through its position at the earliest step, mRNA cap recognition. In many organisms, eIF4E availability is regulated by 4E-binding proteins as well as phosphorylation and sumoylation (Jackson et al., 2010; Xu et al., 2010). However, plants appear to lack 4E-binding proteins, and the role of phosphorylation of eIF4E in translational control is less clear (Pierrat et al., 2007).The eIF4E proteins generally thought to be involved in translation initiation are Class I eIF4E proteins (Joshi et al., 2005), of which two exist in flowering plants: eIF4E, which pairs with eIF4G to form the eIF4F complex, and the plant-specific isoform eIFiso4E, which pairs with eIFiso4G to form eIFiso4F (Mayberry et al., 2011; Patrick and Browning, 2012). Class I eIF4E family members have conserved Trp residues at positions equivalent to Trp-43 and Trp-56 of Homo sapiens eIF4E (Joshi et al., 2005), and the canonical members of this class, such as plant eIF4E and eIFiso4E, have the ability to promote translation through binding of mRNA cap structure and eIF4G (or eIFiso4G).In some organisms, however, secondary Class I isoforms exist with expression patterns and functions divergent from the conserved eIF4E (Rhoads, 2009). Caenorhabditis elegans has four isoforms involved in differentiation between mono- and trimethylated mRNA caps (Keiper et al., 2000) and have specialized roles for regulation of certain sets of mRNAs, particularly in the germline (Amiri et al., 2001; Song et al., 2010). Trypanosoma brucei has four isoforms with varying ability to bind cap analog and eIF4G isoforms (Freire et al., 2011). Schizosaccharomyces pombe has a second eIF4E isoform, eIF4E2, which is nonessential under normal growth conditions, but accumulates in response to high temperatures (Ptushkina et al., 2001). It cannot, however, complement deletion of EIF4E1, and while it can bind capped mRNA and promote translation in vitro, it has reduced ability to bind an eIF4G-derived peptide.Vertebrates encode a novel Class I isoform called EIF4E1B with oocyte-specific expression and functions (Evsikov and Marín de Evsikova, 2009). Zebrafish (Danio rerio) EIF4E1B, with expression limited to muscle and reproductive tissue, has conserved residues identified as necessary for binding cap analog and eIF4G, yet fails to bind either and cannot functionally complement deletion of yeast (Saccharomyces cerevisiae) eIF4E (Robalino et al., 2004). In Xenopus spp. oocytes, the eIF4E1b protein was found to bind eIF4E transporter and cytoplasmic polyadenylation element binding protein to form a translation-repressing complex (Minshall et al., 2007). Drosophila species have undergone extensive expansion of EIF4E-encoding loci to as many as seven different Class I eIF4E isoforms (Tettweiler et al., 2012). The seven EIF4E isoforms of Drosophila melanogaster are differentially expressed, with only five able to bind to eIF4G and complement deletion of yeast eIF4E (Hernández et al., 2005). The eIF4E-3 isoform of D. melanogaster was recently described as having a specific role in spermatogenesis (Hernández et al., 2012).Upon completion of sequencing of the Arabidopsis (Arabidopsis thaliana) genome (Rhee et al., 2003), it was discovered that in addition to the conserved plant EIF4E (At4g18040) and EIFISO4E (At5g35620), there existed a tandem pair of genes of high sequence similarity on chromosome 1 that also encoded Class I eIF4E family proteins, EIF4E1B (At1g29550, also known as EIF4E3) and EIF4E1C (At1g29590, also known as EIF4E2). Published microarray and RNA Sequencing (RNA-Seq) data indicate little to no EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues and enriched in tissues involved in reproduction. The protein sequences contain the residues predicted to be involved in regular eIF4E function but also showed some divergence at highly conserved residues of the canonical plant eIF4E. Genome sequencing data indicate that these genes are part of a divergent eIF4E clade specific to Brassicaceae.The biochemical properties of the eIF4E1b and eIF4E1c proteins were investigated in this work, and it was found that while they can bind mRNA cap analog and eIF4G and support translation in yeast lacking eIF4E, their eIF4G-binding and translation initiation enhancing capabilities in vitro were less robust when compared with the conserved Arabidopsis eIF4E. In addition, it appears that these EIF4E1B-type genes cannot substitute for EIF4E or EIFISO4E in planta because deletion of both of these genes appears to be lethal. Taken together, these findings indicate the EIF4E1B-type genes represent a divergent eIF4E whose roles should be considered separately from the canonical eIF4E in plant translation initiation.     

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34⁺ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34⁺ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34⁺ cells as well as viability and clonogenic assays of CD34⁺ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34⁺ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34⁺ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.