Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response.

The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.

m6A Facilitates eIF4F-Independent mRNA Translation.

In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways.

Genomic dissection of additive and non-additive genetic effects and genomic prediction in an open-pollinated family test of Japanese larch.

Genomic dissection of genetic effects on desirable traits and the subsequent use of genomic selection hold great promise for accelerating the rate of genetic improvement of forest tree species. In this study, a total of 661 offspring trees from 66 open-pollinated families of Japanese larch (Larix kaempferi (Lam.) Carrière) were sampled at a test site. The contributions of additive and non-additive effects (dominance, imprinting and epistasis) were evaluated for nine valuable traits related to growth, wood physical and chemical properties, and competitive ability using three pedigree-based and four Genomics-based Best Linear Unbiased Predictions (GBLUP) models and used to determine the genetic model. The predictive ability (PA) of two genomic prediction methods, GBLUP and Reproducing Kernel Hilbert Spaces (RKHS), was compared. The traits could be classified into two types based on different quantitative genetic architectures: for type I, including wood chemical properties and Pilodyn penetration, additive effect is the main source of variation (38.20-67.46%); for type II, including growth, competitive ability and acoustic velocity, epistasis plays a significant role (50.76-91.26%). Dominance and imprinting showed low to moderate contributions (< 36.26%). GBLUP was more suitable for traits of type I (PAs = 0.37-0.39 vs. 0.14-0.25), and RKHS was more suitable for traits of type II (PAs = 0.23-0.37 vs. 0.07-0.23). Non-additive effects make no meaningful contribution to the enhancement of PA of GBLUP method for all traits. These findings enhance our current understanding of the architecture of quantitative traits and lay the foundation for the development of genomic selection strategies in Japanese larch.

Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

The complete chloroplast genome sequence of Malus × adstringens Zabel 'Hopa' (Rosaceae).

Malus × adstringens Zabel 'Hopa' is an important crabapple cultivar with significant ornamental value. Here, we assembled its complete chloroplast (cp) genome using the next-generation sequencing technology to clarify the phylogenetic relationships in Malus. The total length of the complete chloroplast genome was 160,230 base pairs (bp) with a GC content of 36.50%, consisting of a large single-copy (LSC) region with a sequence length of 88,310 bp, a small single-copy (SSC) region with a sequence length of 19,196 bp, and a pair of inverted repeat (IR) regions of 26,362 bp. The complete chloroplast genome contained 128 genes, namely 84 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. In addition, 73 SSRs were found in the M. 'Hopa' cp genome. The phylogenetic relationship of M. 'Hopa' in Malus is closely related to M. spectabilis (Aiton) Borkh. and then to M. sieversii (Lebed.) M. Roem. Our results demonstrate that it is feasible to resolve the phylogenetic relationships of crabapple cultivars and identify their putative maternal lineages using cp genomic data.

PML/RARalpha fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association.

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.

A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. Start codon-associated ribosomal frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra that are unannotated in the human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational 'noise' in nutrient stress adaptation.

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.

A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. The start codon-associated ribosome frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra unannotated from human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational "noise" in nutrient stress adaptation.

Human SAMD9 is a poxvirus-activatable anticodon nuclease inhibiting codon-specific protein synthesis.

As a defense strategy against viruses or competitors, some microbes use anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNAPhe), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNAPhe depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNAPhe-specific 2'-O-methylation at the wobble position, making virtually all eukaryotic tRNAPhe susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.

Third Generation Genome Sequencing Reveals That Endobacteria in Nematophagous Fungi Esteya vermicola Contain Multiple Genes Encoding for Nematicidal Proteins.

Esteya vermicola is the first recorded endoparasitic nematophagous fungus with high infectivity capacity, attacking the pinewood nematode Bursaphelenchus xylophilus which causes pine wilt disease. Endosymbionts are found in the cytoplasm of E. vermicola from various geographical areas. We sequenced the genome of endobacteria residing in E. vermicola to discover possible biological functions of these widespread endobacteria. Multilocus phylogenetic analyses showed that the endobacteria form a previously unidentified lineage sister to Phyllobacterium myrsinacearum species. The number of genes in the endobacterium was 4542, with 87.8% of the proteins having a known function. It contained a high proportion of repetitive sequences, as well as more Acyl-CoA synthetase genes and genes encoding the electron transport chain, compared with compared with plant-associated P. zundukense Tri 48 and P. myrsinacearum DSM 5893. Thus, this symbiotic bacterium is likely to be more efficient in regulating gene expression and energy release. Furthermore, the endobacteria in nematophagous fungi Esteya vermicola contained multiple nematicidal subtilase/subtilisin encoding genes, so it is likely that endobacteria cooperate with the host to kill nematodes.

Bi-directional ribosome scanning controls the stringency of start codon selection.

The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5'→3' migration of the pre-initiation complex (PIC) along the 5' UTR. In probing translation initiation from ultra-short 5' UTR, we report that an AUG triplet near the 5' end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5' end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5' end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.

Relaxed initiation pausing of ribosomes drives oncogenic translation.

Translation is a crucial process in cancer development and progression. Many oncogenic signaling pathways target the translation initiation stage to satisfy the increased anabolic demands of cancer cells. Using quantitative profiling of initiating ribosomes, we found that ribosomal pausing at the start codon serves as a "brake" to restrain the translational output. In response to oncogenic RAS signaling, the initiation pausing relaxes and contributes to the increased translational flux. Intriguingly, messenger RNA (mRNA) m6A modification in the vicinity of start codons influences the behavior of initiating ribosomes. Under oncogenic RAS signaling, the reduced mRNA methylation leads to relaxed initiation pausing, thereby promoting malignant transformation and tumor growth. Restored initiation pausing by inhibiting m6A demethylases suppresses RAS-mediated oncogenic translation and subsequent tumorigenesis. Our findings unveil a paradigm of translational control that is co-opted by RAS mutant cancer cells to drive malignant phenotypes.

m6A in mRNA coding regions promotes translation via the RNA helicase-containing YTHDC2.

Dynamic mRNA modification in the form of N6-methyladenosine (m6A) adds considerable richness and sophistication to gene regulation. The m6A mark is asymmetrically distributed along mature mRNAs, with approximately 35% of m6A residues located within the coding region (CDS). It has been suggested that methylation in CDS slows down translation elongation. However, neither the decoding feature of endogenous mRNAs nor the physiological significance of CDS m6A has been clearly defined. Here, we found that CDS m6A leads to ribosome pausing in a codon-specific manner. Unexpectedly, removing CDS m6A from these transcripts results in a further decrease of translation. A systemic analysis of RNA structural datasets revealed that CDS m6A positively regulates translation by resolving mRNA secondary structures. We further demonstrate that the elongation-promoting effect of CDS methylation requires the RNA helicase-containing m6A reader YTHDC2. Our findings established the physiological significance of CDS methylation and uncovered non-overlapping function of m6A reader proteins.

Mating patterns and pollen dispersal in a Japanese larch (Larix kaempferi) clonal seed orchard: a case study.

Pollination dynamics highly determines the genetic quality of seed orchard crops. However, there is less research about the effect of mating patterns on seed productivity of orchard crops. So far, clonal seed orchards have been producing genetically improved seedlings used for most Japanese larch (Larix kaempferi (Lamb.) Carr.) plantations in China. In the present study, a total of 17 highly variable simple sequence repeat (SSR) markers were used for genotyping a progeny trial population consisting of 647 open-pollinated progenies germinated from seeds which were collected from 63 maternal clones with 140 potential paternal clones in a Japanese larch clonal seed orchard in China. Paternity analysis was used in the present case study in order to evaluate the level of paternal gametic contribution, estimate pollen contamination and selfing rates, and investigate pollination patterns, pollen dispersal patterns and the impact of mating patterns on seed productivity of orchard crops. We observed 93.7% of the success rate of the parental assignment, unequal paternal gametic contribution (0-12.4%) with 6.3% of the progenies derived from pollen contamination or unsampled pollen donors, and absence of evidence for selfing. We also found that pollination rate highly depended on the distance between pollen donors and maternal parents, the majority of the identified crossing (65.7%) occurred between clones within a 150-m radius, and large variations in growth performance existed among the paternal half-siblings. Progeny growth performance (diameter at breast (DBH) and height (HGT)) was measured at Age-20 in order to investigate the impact of mating patterns on timber production of orchard crops. As either the paternal or maternal, two clones (i. e., clones Z38 and Z62) were identified to have produced progenies with higher average stem volume breeding values than that of all of the progenies. Specifically, the genetic gains for volume were 3.53% for the two clones as paternal parents, and 8.26% as the maternal parents at Age-20. Thus, both elite clones were ideal candidates for the construction of next-generation clonal seed orchards due to their synchronous reproductive phenology with greater crossing rate and higher genetic gain. These results improved the pedigree information to provide solid evidence of mating patterns for future design and effective management of seed orchards and for the development of viable long-term breeding strategies for other coniferous species.

Comparative genomic analyses reveal the features for adaptation to nematodes in fungi.

Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly understood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fungal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphological features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed similarity in large number of transporters and secondary metabolite coding genes. Noteworthy, expanded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theoretical foundation for further research and application.

Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine.

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

Esteya Vermicola, a Nematophagous Fungus Attacking the Pine Wood Nematode, Harbors a Bacterial Endosymbiont Affiliated with Gammaproteobacteria.

Symbioses have played pivotal roles in biological, ecological, and evolutionary diversification. Symbiotic bacteria affect the biology of hosts in a number of ways. Esteya vermicola, an endoparasitic nematophagous fungus, has high infectivity in the pine wood nematode (PWN), which causes devastating ecological damage and economic losses in Asia and Europe. An integration of molecular, phylogenetic, and morphological analyses revealed that surface-sterilized E. vermicola with septate hyphae from different geographic locations harbor bacterial endosymbionts. 16S rRNA gene sequences from four fungal strains all clustered in a well-supported monophyletic clade that was the most closely related to Pseudomonas stutzeri and affiliated with Gammaproteobacteria. The existence and intracellular location of endobacteria was revealed by fluorescent in situ hybridization (FISH). Our results showed that endobacteria were coccoid, vertically inherited, as yet uncultured, and essential symbionts. Ultrastructural observations indicated that young and old endobacteria differed in cell size, cell wall thickness, and the degree of reproduction. The results of the present study provide a fundamental understanding of the endobacteria inside E. vermicola and raise questions regarding the impact of endobacteria on the biology, ecology, and evolution of their fungal host.

The complete mitochondrial genome of nematophagous fungus Esteya vermicola.

The complete mitochondrial genome of the Nematophagous fungus Esteya vermicola CBS 115803 was determined using the PacBio RS II sequencing technology. The circular molecule is 47,282bp in length with a GC content of 24.85%. Annotated genes including 14 conserved protein-coding genes, the large and the small rRNA subunit (rnl and rns) and 27 tRNAs. The phylogenetic analysis showed that E. vermicola had close genetic relationship with the genus Sporothrix.

Constructing a linkage-linkage disequilibrium map using dominant-segregating markers.

The relationship between linkage disequilibrium (LD) and recombination fraction can be used to infer the pattern of genetic variation and evolutionary process in humans and other systems. We described a computational framework to construct a linkage-LD map from commonly used biallelic, single-nucleotide polymorphism (SNP) markers for outcrossing plants by which the decline of LD is visualized with genetic distance. The framework was derived from an open-pollinated (OP) design composed of plants randomly sampled from a natural population and seeds from each sampled plant, enabling simultaneous estimation of the LD in the natural population and recombination fraction due to allelic co-segregation during meiosis. We modified the framework to infer evolutionary pasts of natural populations using those marker types that are segregating in a dominant manner, given their role in creating and maintaining population genetic diversity. A sophisticated two-level EM algorithm was implemented to estimate and retrieve the missing information of segregation characterized by dominant-segregating markers such as single methylation polymorphisms. The model was applied to study the relationship between linkage and LD for a non-model outcrossing species, a gymnosperm species, Torreya grandis, naturally distributed in mountains of the southeastern China. The linkage-LD map constructed from various types of molecular markers opens a powerful gateway for studying the history of plant evolution.