Internal Initiation Site Research Articles

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

Fatigue deformation in a polycrystalline nickel base superalloy at intermediate and high temperature: Competing failure modes

The microstructural configurations that favor early strain localization and fatigue crack initiation at intermediate and high temperature (400 °C–650 °C) have been investigated using novel experimental techniques, including high resolution digital image correlation and transmission scanning electron microscopy. Cyclic fatigue experiments in the high and low cycle fatigue regimes have been performed on a René 88DT polycrystalline nickel-base superalloy at temperatures up to 650 °C and compared to previous fatigue results obtained from tests in the very high cycle fatigue regime. Competing failure modes are observed along with an inversion in the temperature fatigue life dependence of fatigue strength from the low to high cycle fatigue regime. Oxidation-assisted processes are dominant at high applied stresses while cyclic plastic localization and accumulation govern fracture at low applied stresses. In addition, a second competing mode exists in the high and very high cycle fatigue regime from non-metallic inclusions as compared to internal intrinsic initiation sites. The grain-scale features that exhibit strain localization and crack initiation were investigated in detail. Transmission electron microscopy (TEM), transmission scanning electron microscopy (TSEM) and electron channeling contrast imaging have been conducted on samples removed from targeted regions with microstructural configurations that favor crack initiation to characterize the associated dislocation sub-structure and its evolution with temperature. Plasticity is observed to be less localized during cyclic loading at high temperature compared to room temperature. The microstructural features that drive initiation across the temperature range investigated are: twin-parent grains pairs that are at the upper end of the size distribution, are oriented for near maximum elastic modulus mismatch, and have high stresses along planes parallel to the twin boundaries.

Investigation of fatigue crack initiation facets in Ti-6Al-4V using focused ion beam milling and electron backscatter diffraction.

In the very high cycle fatigue regime, internal crack initiation can occur in Ti-6Al-4V because of the formation of facets, which are α grains that have fractured in a transcrystalline and planar manner. Because this crack initiation phase occupies most of the fatigue life, it is essential to understand which mechanisms lead to facet formation. Fatigue tests have been performed on drawn and heat-treated Ti-6Al-4V wires, and the facets at internal crack initiation sites have been analysed in detail in terms of their appearance, their spatial orientation and their crystallographic orientation. The facets were not smooth, but showed surface markings at the nanoscale. In nearly all cases, these markings followed a linear pattern. One anomalous facet, in a sample with the largest grain size, contained a fan-shaped pattern. The facets were at relatively steep angles, mostly between 50° and 70°. Cross-sections of the fracture surfaces have been made by focused ion beam milling and were used to measure the crystallographic orientation of facets by electron backscatter diffraction. Most facet planes coincided with a prismatic lattice plane, and the linear markings were parallel to the prismatic slip direction, which is a strong indication that prismatic slip and slip band formation led to crack initiation. However, the anomalous facet had a near-basal orientation, which points to a possible cleavage mechanism. The cross-sections also exposed secondary cracks, which had formed on prismatic lattice planes, and in some cases early stage facet formation and short crack growth phenomena. The latter observations show that facets can extend through more than one grain, and that there is crack coalescence between facets. The fact that drawn wires have a specific crystallographic texture has led to a different facet formation behaviour compared to what has been suggested in the literature.

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

The chloroplast NAD(P)H dehydrogenase (NDH) C (ndhC) and ndhK genes partially overlap and are cotranscribed in many plants. We previously reported that the tobacco ndhC/K genes are translationally coupled but produce NdhC and NdhK, subunits of the NDH complex, in similar amounts. Generally, translation of the downstream cistron in overlapping mRNAs is very low. Hence, these findings suggested that the ndhK cistron is translated not only from the ndhC 5'UTR but also by an additional pathway. Using an in vitro translation system from tobacco chloroplasts, we report here that free ribosomes enter, with formylmethionyl-tRNA(fMet), at an internal AUG start codon that is located in frame in the middle of the upstream ndhC cistron, translate the 3' half of the ndhC cistron, reach the ndhK start codon, and that, at that point, some ribosomes resume ndhK translation. We detected a peptide corresponding to a 57-amino-acid product encoded by the sequence from the internal AUG to the ndhC stop codon. We propose a model in which the internal initiation site AUG is not designed for synthesizing a functional isoform but for delivering additional ribosomes to the ndhK cistron to produce NdhK in the amount required for the assembly of the NDH complex. This pathway is a unique type of translation to produce protein in the needed amount with the cost of peptide synthesis.

Microstructure investigations on two different aluminum wrought alloys after very high cycle fatigue

Fatigue studies were conducted under load control with a servo-hydraulic testing machine with smooth specimens of the aluminum wrought alloys EN AW-6056-T6 (Rm=399MPa) and EN AW-6082-T5 (Rm=356MPa) up to a maximum number of N=2×108 and 109cycles, respectively. The results show for both aluminum alloys that the fatigue strength decreases with increasing number of cycles after the kneepoint of the S–N-curve and that approximately at this point a transition of the crack initiation site from the surface to the subsurface occurs. All fractured specimens were investigated with a scanning electron microscope (SEM). Large defects like primary intermetallic particles could not be found at the crack initiation sites. The difference between the subsurface non-defect crack initiation sites of both alloys is to be found in the fractographic structure. The internal crack initiation site of EN AW-6056-T6 shows a multiplicity of cleavage-like planes contrary to the flat area found at the alloy EN AW-6082-T5. These results were already presented in [1]. The scope of this present paper is new microstructural investigations and the formulation of a failure model which respects the results of different scales of microscopy. Electron back scatter diffraction analysis (EBSD analysis) and transmission electron microscopy (TEM) were done for both alloys to characterize the microstructure and to compare unstressed and tested materials. These differences are discussed with regard to the differences in failure mechanisms.

Determinants of Translation Elongation Speed and Ribosomal Profiling Biases in Mouse Embryonic Stem Cells

Ribosomal profiling is a promising approach with increasing popularity for studying translation. This approach enables monitoring the ribosomal density along genes at a resolution of single nucleotides.In this study, we focused on ribosomal density profiles of mouse embryonic stem cells. Our analysis suggests, for the first time, that even in mammals such as M. musculus the elongation speed is significantly and directly affected by determinants of the coding sequence such as: 1) the adaptation of codons to the tRNA pool; 2) the local mRNA folding of the coding sequence; 3) the local charge of amino acids encoded in the codon sequence. In addition, our analyses suggest that in general, the translation velocity of ribosomes is slower at the beginning of the coding sequence and tends to increase downstream.Finally, a comparison of these data to the expected biophysical behavior of translation suggests that it suffers from some unknown biases. Specifically, the ribosomal flux measured on the experimental data increases along the coding sequence; however, according to any biophysical model of ribosomal movement lacking internal initiation sites, the flux is expected to remain constant or decrease. Thus, developing experimental and/or statistical methods for understanding, detecting and dealing with such biases is of high importance.

Precise control of surface physicochemical properties for electrospun fiber mats by surface-initiated radical polymerization

Non-woven fiber mats were fabricated with the electrospinning method using poly(methyl methacrylate)-co-poly(2-(2-bromoisobutyryloxy)ethyl methacrylate) in various solvents. The surface morphology of the electrospun fibers depended on the solvent vapor pressure and polymer concentration. Surface-initiated atom transfer radical polymerization (ATRP) with 3-(N-2-methacryloyloxyethyl-N,N-dimethyl) ammonatopropanesulfonate), 2-hydroxyethyl methacrylate or 2-(perfluorooctyl)ethyl acrylate generated surface-grafted polymers, as determined by X-ray photoelectron spectroscopy. Scanning electron microscopic observation revealed that the apparent fiber morphology did not change upon modification. The atomic force microscopic images of the grafted fiber cross-sections indicated that monomers and solvents penetrated slightly into the fibers and polymerization occurred at both internal and external initiation sites. Physicochemical properties, such as contact angle, hydrophobicity and hydrophilicity, and wettability, could be altered by the proper selection of substrates (spin-coated flat films or the variously prepared non-woven fiber mats). We have successfully prepared hydrophilic and hydrophobic fiber surfaces by a combination of the electrospinning protocol and surface-initiated ATRP. Functional non-woven fiber mats were prepared by combining the electrospinning method and surface-initiated atom transfer radical polymerization (ATRP). The surface-initiated ATRP of 3-(N-2-methacryloyloxyethyl-N,N-dimethyl) ammonatopropanesulfonate), 2-hydroxyethyl methacrylate and 2-(perfluorooctyl)ethyl acrylate proceeded successfully from the fiber surface of poly(methyl methacrylate)-co-poly(2-(2-bromoisobutyryloxy)ethyl methacrylate) without any structural changes. A grafted layer after the surface-initiated ATRP was observed by atomic force microscopy. Static contact angles against water were also evaluated to discuss the relationship between the roughness of the electrospun non-woven fiber mats and wettability.

Widespread remodeling of mid-coding sequence nucleosomes by Isw1

In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5’ regions of genes and may regulate transcriptional elongation.

Les récepteurs mitochondriaux de la triiodothyronine : import et mécanismes d'action

Thyroid hormone exerts a diversity of physiological influences over developmental and metabolic processes. Searching for receptors able to mediate this extended regulation led to the identification of triiodothyronine (T3) nuclear receptors encoded by two different genes, c-erbA alpha (TR alpha) and c-erbA beta (TR beta). More recently, two N-terminally truncated forms of the triiodothyronine nuclear receptor TR alpha 1, with molecular weights of 43 and 28 kDa, have been discovered in mitochondria. Synthesized through the use of internal initiation sites of translation occurring in the TR alpha 1 transcript, they are addressed into mitochondria according to an atypical process. Two mitochondrial import sequences have been characterized in the C-terminal part of these proteins; in addition, their N-terminal part, devoid of negative charges, plays a permissive role in this import. Whereas the function of p28 remains unknown, p43 is a T3-dependent transcription factor of the mitochondrial genome, acting through dimeric complexes involving at least two other truncated forms of nuclear receptors, mtRXR and mtPPAR. P43 activation by T3 stimulates mitochondrial protein synthesis, respiratory chain activity and mitochondriogenesis. Through the mitochondrial/nuclear crosstalk, this direct T3 mitochondrial pathway influences the expression of nuclear genes involved in the regulation of cell proliferation and differentiation. In particular, in myoblasts, p43 overexpression stimulates terminal differentiation and induces a preferential expression of slow myosin, by down-regulating c-Myc expression and up-regulating calcineurin and myogenin expression. Comparison of the respective influences of the nuclear and mitochondrial T3 pathways demonstrates either both additivity (myoblast differentiation), complementarity (mitochondriogenesis, myoblast differentiation) or opposite influences (myosin expression), thus indicating that these two pathways introduce a fine-tuning of the hormone influence.

ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Effect of microstructure on ultra-high cycle fatigue behavior of Ti–6Al–4V

The fatigue behavior of Ti–6Al–4V alloy with the bimodal and basketweave microstructures has been investigated. The results show that the S– N curves of Ti–6Al–4V with both microstructures continuously decrease with increasing the number of cycles to failure and have no horizontal asymptote in the regime of 10 5 to 10 9 cycles. SEM observation of fracture surface indicates that the crack initiation sites abruptly shift from surface to interior of the specimen with decreasing the stress amplitude. It is found that the surface crack initiation mainly results from the machining traces. Nevertheless, most of the internal fatigue cracks initiate in the border region of primary α-grains in bimodal microstructure or at α–β interfaces in basketweave microstructure, which induced by inhomogeneity of microstructures and incompatibility of deformation between two phases. The size of the internal crack initiation site was evaluated and the dependence of the fatigue life on the location and size of the initiation sites is discussed.

Dominant-interfering C/EBPα stimulates primitive erythropoiesis in zebrafish

We investigated the role of CCAAT enhancer-binding protein-alpha (C/EBPalpha) during zebrafish embryonic blood development. Whole-mount mRNA in situ hybridization was performed to determine the spatio-temporal expression pattern of zebrafish cebpa in developing hematopoietic progenitors. A deletion mutation of cebpa (zD420), which mimics the human dominant-negative mutations of C/EBPalpha, was transfected into CV1 cell line to evaluate its transcriptional activity in vitro and injected into zebrafish embryos at the one- to two-cell stage to examine its effects on primitive hematopoiesis during early zebrafish development. Zebrafish cebpa is expressed in the anterior and posterior lateral plate mesoderm at 12 hours postfertilization, along with scl, pu.1, and gata1 in developing hematopoietic progenitors. In vitro, the deletion mutation of cebpa (zD420) prevents expression of the full-length protein, allowing the expression of truncated isoforms from internal translational initiation sites. As in the human, the truncated zebrafish C/EBPalpha proteins did not activate the expression of known target granulocytic genes, and in fact suppressed transactivation that was induced in vitro by the full-length protein. Forced expression of the zD420 mRNA in zebrafish embryos led to an expansion of primitive erythropoiesis, without a discernible effect on granulopoiesis. Expression of the truncated isoforms of cebpa alters the developmental pattern of hematopoietic progenitor cells during embryogenesis.

Fatigue Strength of Alloy 718 at High-Temperatures

The high-temperature fatigue properties of an Alloy 718 were investigated up to 10 7 cycles. Combined with the data for base metals and welded joints, the relationship between stress amplitude and number of cycles to failure (fatigue life) was characterized by two steps in the S-N curve. The fracture mechanisms were different between high and low stress levels. At high stress levels, fatigue fractures initiated from the specimen surface, whereas at low stress levels, the initiation was inside the surface. The internal fracture initiation sites were crystallographic flat facets of austenitic grains.

Fatigue Characteristics of Low Cost β Titanium Alloys for Healthcare and Medical Applications

Two new low cost β titanium alloys, Ti–4.3Fe–7.1Cr (TFC alloy) and Ti–4.3Fe–7.1Cr–3.0Al (TFCA alloy) for healthcare and medical applications have been recently developed. As for such applications, the alloys are necessary to have high fatigue performance. The aim of this study is, therefore, to investigate fatigue characteristics of the alloys subjected to solution treatment above β transus. Fatigue tests were carried out at a stress ratio, R, of 0.1 and a frequency of 10 Hz.Fatigue limit of the solution treated TFC alloy is higher than that of the solution treated TFCA alloy, but both are higher than that of the existing biometallic materials. Fatigue strength of the TFC alloy is almost independent of solution treatment temperature, while, fatigue strength of the TFCA alloy strongly depends on solution treatment temperature, especially, in the low cycle fatigue life (LCF) region. The fatigue ratio and biofunctionality of these new alloys are much higher than those of the existing biometallic materials. In general, a crack initiates from the surface in the LCF region and from subsurface (internal) in the high cycle fatigue life (HCF) region for the TFC alloy, while, in the case of the TFCA alloy, a crack tends to initiate from the subsurface in both LCF and HCF regions. The internal crack initiation sites are found to be the area with low β phase stability in the LCF region and at the area with high stability of β phase in the HCF region. The relatively low fatigue strength of TFCA alloy is associated with the addition of Al that leads to precipitate α phase in which both crack initiation and facet formation are easier to occur.

TRNA-like structure regulates translation of Brome mosaic virus RNA.

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

Role of an internal ribosome entry site in the translational control of the human transcription factor Sp3

Sp1 and Sp3 are transcription factors involved in the regulation of numerous genes involved in oncogenesis. Sp3 is a bi-functional transcription factor with three different isoforms. Its bi-functional activity may in part be regulated by the relative expression of these isoforms. Northern blot analysis of Sp3 detects only a single transcript. Analysis of the known Sp3 cDNA sequence shows a high GC content and seven out-of-frame AUG codons located between the 5'-end of the mRNA and the two internal AUG initiation sites. This makes it highly unlikely that cap-recruited, translation initiation competent ribosomes could reach the internal start sites. A full human Sp3 expression construct was cloned. A bicistronic vector using Renilla and firefly luciferase showed internal ribosome entry site (IRES) activity in Sp3 RNA immediately 5' to the internal AUG sites. Also, the two smaller isoforms were translated more efficiently when full-length, uncapped transcripts were used, while the larger isoform was not translated. Mutants of Sp3 with AUG codons introduced 5' of the two internal start sites were generated. Results showed that they were unable to suppress the smaller isoforms in vitro. Furthermore, dual non-AUG to AUG mutations showed occlusion of the second introduced isoform (i.e., the isoform situated more 3') but not of the internally initiated isoforms. These experiments are consistent with IRES-mediated translation of the two smaller isoforms of Sp3. The presence of an IRES allows the possibility that Sp3 isoform ratios and activity are controlled at the translational level. This mechanism may allow cells to control the expression of numerous genes during mitosis and, thus, have profound effects on cell cycle regulation and tumorigenesis.

Internal initiation sites of de novo RNA synthesis within the hepatitis C virus polypyrimidine tract

A variety of 3 ′-untranslated regions (UTRs) were cloned from infectious hepatitis C virus human samples and examined in NS5B polymerase de novo initiation reactions. We isolated and characterized four distinct 3 ′-UTRs that harbor the conserved terminal 98 nucleotides, but have poly(U/UC) tracts of 25, 93, 98, and 101 nucleotides, respectively. Reconstitution of de novo initiation by the mature NS5B with the different 3 ′-UTR RNA substrates revealed distinctively sized products that are consistent with internal initiation at specific sites within the polypyrimidine tract. These sites were further mapped by demonstrating that nucleotide substitutions of the cytidylate stretches within the poly(U/UC) template eliminate specific products of de novo synthesis and by showing that these products could be radiolabeled by [γ- 32P]GTP. We also examine analogs that can substitute for GTP in this reaction.

Internal Initiation by the Cucumber Necrosis Virus RNA-Dependent RNA Polymerase Is Facilitated by Promoter-like Sequences

Tombusviruses, small positive sense RNA viruses of plants, are replicated by the viral-coded RNA-dependent RNA polymerase (RdRp) in infected cells. An unusual feature of the tombusvirus RdRp that is partially purified from cucumber necrosis virus (CNV)-infected plants is the ability to initiate complementary RNA synthesis from several internal positions on minus-strand templates derived from DI RNAs (19). In this study, we used template deletion, mutagenesis, and oligo-based inhibition of RNA synthesis to map the internal initiation sites observed with the in vitro CNV RdRp system. Comparing sequences around the internal initiation sites reveals that they have either (i) similar sequences to the core minus-strand initiation promoter; or (ii) similar structures to the core plus-strand initiation promoter. In addition, we find similarities among the internal initiation sites and the subgenomic RNA initiation sites. These similarities suggest that the mechanism of internal initiation is similar to initiation from the terminal core promoters or the putative subgenomic promoter sequences. We propose that internal initiation on full-length RNA templates may be important in defective interfering (DI) RNA formation/evolution by producing intermediate templates for RNA recombination in tombusviruses. This may explain why tombusviruses are frequently associated with DI RNAs.

Release factor 2 frameshifting sites in different bacteria.

The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the approximately 70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine-Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure.

Effects of mercuric chloride on the regulation of expression of the acute phase response components α 1-acid glycoprotein and C/EBP transcription factors

We have previously shown that in response to treatment with HgCl 2, the adult mouse liver exhibits both transcriptional and translational regulation of the acute phase response genes. In this study we asked whether the heavy metal treatment affects the regulation of the C/EBP transcription factors which play a key role in regulation of the acute phase response gene. Our studies have shown that the AGP gene is transcriptionally activated while transcription of the CCAAT/enhancer-binding trans-activating protein (C/EBP)α gene is slightly down-regulated and that of the C/EBPβ gene does not respond. Both the C/EBPα and C/EBPβ mRNAs produce multiple isoforms possibly by alternative translation initiation (ATI) of multiple internal AUG initiation sites. The C/EBPβ mRNA appears to be stabilized. Although similar regulatory processes occur in response HgCl 2 vs. LPS, our data suggest that the translational processes (ATI) are differentially affected. In addition, a major difference lies in the fact that the C/EBPβ gene is not transcriptionally activated by HgCl 2. Our data show decreased binding activity and pool levels of the C/EBPα isoform (p42 C/EBPα) and increased binding activity and pool levels of C/EBPβ isoform (p35 C/EBPβ) in response to HgCl 2. We propose that this isoform may be involved in the regulation of AGP gene expression in response to heavy metals and that there is a significant difference between the HgCl 2-mediated and LPS-mediated inflammatory response.