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Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids. Author summary: Conventional automatic genome annotation algorithms often neglect open reading frames smaller than 300 nucleotides (sORF). There are several reasons hindering automatic annotation and prediction of short genes: (i) sORFs possess insufficient sequence information for domain and homology search, (ii) only a limited number of experimentally validated sORFs can serve as templates, and (iii) sORFs show the tendency to be species-specific. We thus established a proteogenomics workflow, which is executed by two open source tools, SALT and Pepper (https://gitlab.com/s.fuchs/pepper), and uses peptide data obtained by mass spectrometry for identification of genes in bacteria that are hardly predictable by automatic annotation algorithms. As a proof of concept, we selected Staphylococcus aureus, one of the most frequently sequenced bacteria and identified 36 proteins not yet considered in the used genome annotation of S. aureus Newman. 24 there of are novel small proteins with up to 100 aa (SP100) in S. aureus Newman. This clearly demonstrates that our workflow is ideally suited to improve gene annotation of already annotated bacterial genomes. In the future, it may also facilitate protein and ORF detection in not annotated bacterial genomes. [ABSTRACT FROM AUTHOR]

Double-stranded RNA (dsRNA) is produced both by virus and host. Its recognition by the melanoma differentiation–associated gene 5 (MDA5) initiates type I interferon responses. How can a host distinguish self-transcripts from nonself to ensure that responses are targeted correctly? Here, I discuss a role for MDA5 helicase in inducing Z-RNA formation by Alu inverted repeat (AIR) elements. These retroelements have highly conserved sequences that favor Z-formation, creating a site for the dsRNA-specific deaminase enzyme ADAR1 to dock. The subsequent editing destabilizes the dsRNA, ending further interaction with MDA5 and terminating innate immune responses directed against self. By enabling self-recognition, Alu retrotransposons, once invaders, now are genetic elements that keep immune responses in check. I also discuss the possible but less characterized roles of the other helicases in modulating innate immune responses, focusing on DExH-box helicase 9 (DHX9) and Mov10 RISC complex RNA helicase (MOV10). DHX9 and MOV10 function differently from MDA5, but still use nucleic acid structure, rather than nucleotide sequence, to define self. Those genetic elements encoding the alternative conformations involved, referred to as flipons, enable helicases to dynamically shape a cell's repertoire of responses. In the case of MDA5, Alu flipons switch off the dsRNA-dependent responses against self. I suggest a number of genetic systems in which to study interactions between flipons and helicases further. [ABSTRACT FROM AUTHOR]

Phosphohexomutase superfamily enzymes catalyze the reversible intramolecular transfer of a phosphoryl moiety on hexose sugars. Bacillus subtilis phosphoglucomutase PgcA catalyzes the reversible interconversion of glucose 6-phosphate (Glc-6-P) and glucose 1-phosphate (Glc-1-P), a precursor of UDP-glucose (UDP-Glc). B. subtilis phosphoglucosamine mutase (GlmM) is a member of the same enzyme superfamily that converts glucosamine 6-phosphate (GlcN-6-P) to glucosamine 1-phosphate (GlcN-1-P), a precursor of the amino sugar moiety of peptidoglycan. Here, we present evidence that B. subtilis PgcA possesses activity as a phosphoglucosamine mutase that contributes to peptidoglycan biosynthesis. This activity was made genetically apparent by the synthetic lethality of pgcA with glmR, a positive regulator of amino sugar biosynthesis, which can be specifically suppressed by overproduction of GlmM. A gain-of-function mutation in a substrate binding loop (PgcA G47S) increases this secondary activity and suppresses a glmR mutant. Our results demonstrate that bacterial phosphoglucomutases may possess secondary phosphoglucosamine mutase activity, and that this dual activity may provide some level of functional redundancy for the essential peptidoglycan biosynthesis pathway. [ABSTRACT FROM AUTHOR]

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington’s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis. [ABSTRACT FROM AUTHOR]

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers. [ABSTRACT FROM AUTHOR]

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability. [ABSTRACT FROM AUTHOR]

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome. [ABSTRACT FROM AUTHOR]

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA’s 3’ UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs interact to regulate gene expression is likely extensive, we have only begun to unravel the mechanisms of this functional cooperation. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK-1, in modulating miRNA activity during C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 plays a role in processing of some but not all miRNAs and is not required for ALG-1/AIN-1 miRISC assembly. We suggest that HRPK-1 may functionally interact with miRNAs by both affecting miRNA processing and by enhancing miRNA/miRISC gene regulatory activity and present models for its activity. [ABSTRACT FROM AUTHOR]

The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. Here, we studied the consequences of modulating iron metabolism on the growth of Drosophila imaginal discs. We find that reducing the levels of the ferritin heavy chain in the larval wing discs leads to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. We propose that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation. [ABSTRACT FROM AUTHOR]

Stressful life events are major environmental risk factors for anxiety disorders, although not all individuals exposed to stress develop clinical anxiety. The molecular mechanisms underlying the influence of environmental effects on anxiety are largely unknown. To identify biological pathways mediating stress-related anxiety and resilience to it, we used the chronic social defeat stress (CSDS) paradigm in male mice of two inbred strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), that differ in their susceptibility to stress. Using a multi-omics approach, we identified differential mRNA, miRNA and protein expression changes in the bed nucleus of the stria terminalis (BNST) and blood cells after chronic stress. Integrative gene set enrichment analysis revealed enrichment of mitochondrial-related genes in the BNST and blood of stressed mice. To translate these results to human anxiety, we investigated blood gene expression changes associated with exposure-induced panic attacks. Remarkably, we found reduced expression of mitochondrial-related genes in D2 stress-susceptible mice and in exposure-induced panic attacks in humans, but increased expression of these genes in B6 stress-susceptible mice. Moreover, stress-susceptible vs. stress-resilient B6 mice displayed more mitochondrial cross-sections in the post-synaptic compartment after CSDS. Our findings demonstrate mitochondrial-related alterations in gene expression as an evolutionarily conserved response in stress-related behaviors and validate the use of cross-species approaches in investigating the biological mechanisms underlying anxiety disorders. [ABSTRACT FROM AUTHOR]

Iran, despite its size, geographic location and past cultural influence, has largely been a blind spot for human population genetic studies. With only sparse genetic information on the Iranian population available, we pursued its genome-wide and geographic characterization based on 1021 samples from eleven ethnic groups. We show that Iranians, while close to neighboring populations, present distinct genetic variation consistent with long-standing genetic continuity, harbor high heterogeneity and different levels of consanguinity, fall apart into a cluster of similar groups and several admixed ones and have experienced numerous language adoption events in the past. Our findings render Iran an important source for human genetic variation in Western and Central Asia, will guide adequate study sampling and assist the interpretation of putative disease-implicated genetic variation. Given Iran’s internal genetic heterogeneity, future studies will have to consider ethnic affiliations and possible admixture. [ABSTRACT FROM AUTHOR]

During vertebrate early embryogenesis, the ventral development is directed by the ventral-to-dorsal activity gradient of the bone morphogenetic protein (BMP) signaling. As secreted ligands, the extracellular traffic of BMP has been extensively studied. However, it remains poorly understood that how BMP ligands are secreted from BMP-producing cells. In this work, we show the dominant role of Marcksb controlling the secretory process of Bmp2b via interaction with Hsp70 in vivo. We firstly carefully characterized the role of Marcksb in promoting BMP signaling during dorsoventral axis formation through knockdown approach. We then showed that Marcksb cell autonomously regulates the trafficking of Bmp2b from producing cell to the extracellular space and both the total and the extracellular Bmp2b was decreased in Marcksb-deficient embryos. However, neither the zygotic mutant of marcksb (Zmarcksb) nor the maternal zygotic mutant of marcksb (MZmarcksb) showed any defects of dorsalization. In contrast, the MZmarcksb embryos even showed increased BMP signaling activity as measured by expression of BMP targets, phosphorylated Smad1/5/9 levels and imaging of Bmp2b, suggesting that a phenomenon of “genetic over-compensation” arose. Finally, we revealed that the over-compensation effects of BMP signaling in MZmarcksb was achieved through a sequential up-regulation of MARCKS-family members Marcksa, Marcksl1a and Marcksl1b, and MARCKS-interacting protein Hsp70.3. We concluded that the Marcksb modulates BMP signaling through regulating the secretory pathway of Bmp2b. [ABSTRACT FROM AUTHOR]

In mammals, dosage compensation of sex chromosomal genes between females (XX) and males (XY) is achieved through X-chromosome inactivation (XCI). The X-linked X-inactive-specific transcript (Xist) long noncoding RNA is indispensable for XCI and initiates the process early during development by spreading in cis across the X chromosome from which it is transcribed. During XCI, Xist RNA triggers gene silencing, recruits a plethora of chromatin modifying factors, and drives a major structural reorganization of the X chromosome. Here, we review our knowledge of the multitude of epigenetic events orchestrated by Xist RNA to allow female mammals to survive through embryonic development by establishing and maintaining proper dosage compensation. In particular, we focus on recent studies characterizing the interaction partners of Xist RNA, and we discuss how they have affected the field by addressing long-standing controversies or by giving rise to new research perspectives that are currently being explored. This review is dedicated to the memory of Denise Barlow, pioneer of genomic imprinting and functional long noncoding RNAs (lncRNAs), whose work has revolutionized the epigenetics field and continues to inspire generations of scientists. [ABSTRACT FROM AUTHOR]

Errors throughout gene expression are likely deleterious, hence genomes are under selection to ameliorate their consequences. Additional stop codons (ASCs) are in-frame nonsense ‘codons’ downstream of the primary stop which may be read by translational machinery should the primary stop have been accidentally read through. Prior evidence in several eukaryotes suggests that ASCs are selected to prevent potentially-deleterious consequences of read-through. We extend this evidence showing that enrichment of ASCs is common but not universal for single cell eukaryotes. By contrast, there is limited evidence as to whether the same is true in other taxa. Here, we provide the first systematic test of the hypothesis that ASCs act as a fail-safe mechanism in eubacteria, a group with high read-through rates. Contra to the predictions of the hypothesis we find: there is paucity, not enrichment, of ASCs downstream; substitutions that degrade stops are more frequent in-frame than out-of-frame in 3’ sequence; highly expressed genes are no more likely to have ASCs than lowly expressed genes; usage of the leakiest primary stop (TGA) in highly expressed genes does not predict ASC enrichment even compared to usage of non-leaky stops (TAA) in lowly expressed genes, beyond downstream codon +1. Any effect at the codon immediately proximal to the primary stop can be accounted for by a preference for a T/U residue immediately following the stop, although if anything, TT- and TC- starting codons are preferred. We conclude that there is no compelling evidence for ASC selection in eubacteria. This presents an unusual case in which the same error could be solved by the same mechanism in eukaryotes and prokaryotes but is not. We discuss two possible explanations: that, owing to the absence of nonsense mediated decay, bacteria may solve read-through via gene truncation and in eukaryotes certain prion states cause raised read-through rates. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is currently one of the most important challenges to the treatment of bacterial infections. A critical issue to combat AMR is to restrict its spread. In several instances, bacterial plasmids are involved in the global spread of AMR. Plasmids belonging to the incompatibility group (Inc)HI are widespread in Enterobacteriaceae and most of them express multiple antibiotic resistance determinants. They play a relevant role in the recent spread of colistin resistance. We present in this report novel findings regarding IncHI plasmid conjugation. Conjugative transfer in liquid medium of an IncHI plasmid requires expression of a plasmid-encoded, large-molecular-mass protein that contains an Ig-like domain. The protein, termed RSP, is encoded by a gene (ORF R0009) that maps in the Tra2 region of the IncHI1 R27 plasmid. The RSP protein is exported outside the cell by using the plasmid-encoded type IV secretion system that is also used for its transmission to new cells. Expression of the protein reduces cell motility and enables plasmid conjugation. Flagella are one of the cellular targets of the RSP protein. The RSP protein is required for a high rate of plasmid transfer in both flagellated and nonflagellated Salmonella cells. This effect suggests that RSP interacts with other cellular structures as well as with flagella. These unidentified interactions must facilitate mating pair formation and, hence, facilitate IncHI plasmid conjugation. Due to its location on the outer surfaces of the bacterial cell, targeting the RSP protein could be a means of controlling IncHI plasmid conjugation in natural environments or of combatting infections caused by AMR enterobacteria that harbor IncHI plasmids. [ABSTRACT FROM AUTHOR]

Wnt proteins are secreted signaling factors that regulate cell fate specification and patterning decisions throughout the animal kingdom. In the Drosophila wing epithelium, Wingless (Wg, the homolog of Wnt1) is secreted from a narrow strip of cells at the dorsal-ventral boundary. However, the route of Wg secretion in polarized epithelial cells remains poorly understood and key proteins involved in this process are still unknown. Here, we performed an in vivo RNAi screen and identified members of the exocyst complex to be required for apical but not basolateral Wg secretion. Specifically blocking the apical Wg secretion leads to reduced downstream signaling. Using an in vivo ‘temporal-rescue’ assay, our results further indicate that apically secreted Wg activates target genes that require high signaling activity. In conclusion, our results demonstrate that the exocyst is required for an apical route of Wg secretion from polarized wing epithelial cells. [ABSTRACT FROM AUTHOR]

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins. [ABSTRACT FROM AUTHOR]

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants. [ABSTRACT FROM AUTHOR]

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes. [ABSTRACT FROM AUTHOR]

Telomere shortening is associated with aging and age-associated diseases. Additionally, telomere dysfunction resulting from telomerase gene mutation can lead to premature aging, such as apparent skin atrophy and hair loss. However, the molecular signaling linking telomere dysfunction to skin atrophy remains elusive. Here we show that dysfunctional telomere disrupts BMP/pSmad/P63 signaling, impairing epidermal stem cell specification and differentiation of skin and hair follicles. We find that telomere shortening mediated by Terc loss up-regulates Follistatin (Fst), inhibiting pSmad signaling and down-regulating P63 and epidermal keratins in an ESC differentiation model as well as in adult development of telomere-shortened mice. Mechanistically, short telomeres disrupt PRC2/H3K27me3-mediated repression of Fst. Our findings reveal that skin atrophy due to telomere dysfunction is caused by a previously unappreciated link with Fst and BMP signaling that could be explored in the development of therapies. [ABSTRACT FROM AUTHOR]

In all kingdoms of life, is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by prior to cell division to ensure each daughter cell receives the full complement of . DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life. [ABSTRACT FROM AUTHOR]

DNA methylation and epigenetic silencing play important roles in the regulation of transposable elements (TEs) in many eukaryotic genomes. A majority of the maize genome is derived from TEs that can be classified into different orders and families based on their mechanism of transposition and sequence similarity, respectively. TEs themselves are highly methylated and it can be tempting to view them as a single uniform group. However, the analysis of DNA methylation profiles in flanking regions provides evidence for distinct groups of chromatin properties at different TE families. These differences among TE families are reproducible in different tissues and different inbred lines. TE families with varying levels of DNA methylation in flanking regions also show distinct patterns of chromatin accessibility and modifications within the TEs. The differences in the patterns of DNA methylation flanking TE families arise from a combination of non-random insertion preferences of TE families, changes in DNA methylation triggered by the insertion of the TE and subsequent selection pressure. A set of nearly 70,000 TE polymorphisms among four assembled maize genomes were used to monitor the level of DNA methylation at haplotypes with and without the TE insertions. In many cases, TE families with high levels of DNA methylation in flanking sequence are enriched for insertions into highly methylated regions. The majority of the >2,500 TE insertions into unmethylated regions result in changes in DNA methylation in haplotypes with the TE, suggesting the widespread potential for TE insertions to condition altered methylation in conserved regions of the genome. This study highlights the interplay between TEs and the methylome of a major crop species. [ABSTRACT FROM AUTHOR]

Pancreatic adenocarcinoma (PC) is a lethal malignancy that is familial or associated with genetic syndromes in 10% of cases. Gene-based surveillance strategies for at-risk individuals may improve clinical outcomes. However, familial PC (FPC) is plagued by genetic heterogeneity and the genetic basis for the majority of FPC remains elusive, hampering the development of gene-based surveillance programs. The study was powered to identify genes with a cumulative pathogenic variant prevalence of at least 3%, which includes the most prevalent PC susceptibility gene, BRCA2. Since the majority of known PC susceptibility genes are involved in DNA repair, we focused on genes implicated in these pathways. We performed a region-based association study using the Mixed-Effects Score Test, followed by leave-one-out characterization of PC-associated gene regions and variants to identify the genes and variants driving risk associations. We evaluated 398 cases from two case series and 987 controls without a personal history of cancer. The first case series consisted of 109 patients with either FPC (n = 101) or PC at ≤50 years of age (n = 8). The second case series was composed of 289 unselected PC cases. We validated this discovery strategy by identifying known pathogenic BRCA2 variants, and also identified SMG1, encoding a serine/threonine protein kinase, to be significantly associated with PC following correction for multiple testing (p = 3.22x10-7). The SMG1 association was validated in a second independent series of 532 FPC cases and 753 controls (p<0.0062, OR = 1.88, 95%CI 1.17–3.03). We showed segregation of the c.4249A>G SMG1 variant in 3 affected relatives in a FPC kindred, and we found c.103G>A to be a recurrent SMG1 variant associating with PC in both the discovery and validation series. These results suggest that SMG1 is a novel PC susceptibility gene, and we identified specific SMG1 gene variants associated with PC risk. [ABSTRACT FROM AUTHOR]

Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held view across the broad community of life scientists that circular genome-sized molecules are the primary form of plant mitochondrial DNA, despite the understanding by plant mitochondrial researchers that this is an inaccurate and outdated concept. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative arrangements (isoforms). Most mitochondrial genomes have been assembled using methods unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. We characterized a comprehensive, complex set of isoforms within each species and compared genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms in our data, we infer that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other Lactuca species can be largely explained by rare recombination events that rearranged the structure. Our data demonstrate that representations of plant mitochondrial genomes as simple, circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms. [ABSTRACT FROM AUTHOR]

Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions. [ABSTRACT FROM AUTHOR]

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway. [ABSTRACT FROM AUTHOR]

The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5/ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5/ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications. [ABSTRACT FROM AUTHOR]

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs. [ABSTRACT FROM AUTHOR]

The synaptonemal complex (SC) is a proteinaceous scaffold required for synapsis and recombination between homologous chromosomes during meiosis. Although the SC has been linked to differences in genome-wide crossover rates, the genetic basis of standing variation in SC structure remains unknown. To investigate the possibility that recombination evolves through changes to the SC, we characterized the genetic architecture of SC divergence on two evolutionary timescales. Applying a novel digital image analysis technique to spermatocyte spreads, we measured total SC length in 9,532 spermatocytes from recombinant offspring of wild-derived mouse strains with differences in this fundamental meiotic trait. Using this large dataset, we identified the first known genomic regions involved in the evolution of SC length. Distinct loci affect total SC length divergence between and within subspecies, with the X chromosome contributing to both. Joint genetic analysis of MLH1 foci—immunofluorescent markers of crossovers—from the same spermatocytes revealed that two of the identified loci also confer differences in the genome-wide recombination rate. Causal mediation analysis suggested that one pleiotropic locus acts early in meiosis to designate crossovers prior to SC assembly, whereas a second locus primarily shapes crossover number through its effect on SC length. One genomic interval shapes the relationship between SC length and recombination rate, likely modulating the strength of crossover interference. Our findings pinpoint SC formation as a key step in the evolution of recombination and demonstrate the power of genetic mapping on standing variation in the context of the recombination pathway. [ABSTRACT FROM AUTHOR]

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these “hungry spliceosome” conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of “intronization”, whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function. [ABSTRACT FROM AUTHOR]

The role of long non-coding RNA (lncRNA) in the progression of Nasopharyngeal carcinoma (NPC) has not been fully elucidated. The study was designed to explore the functional role of NKILA, a newly identified lncRNA, in the progression of NPC. We performed a lncRNA expression profile microarray using four NPC and paired para-cancerous tissues. NKILA was identified as a potential functional lncRNA by this lncRNA expression profile. We used 107 paraffin-embedded NPC tissues with different TNM stages to detect the expression of NKILA and analyzed the survival data by Log-rank test and Cox regression. The role of NKILA and its underlying mechanisms in the progression of NPC were evaluated by a series of experiments in vitro and vivo by silencing or expressing NKILA. Compared with control tissues, NKILA expression was identified to be decreased in NPC tissues. Low NKILA expression was correlated with unfavorable clinicopathological features and predicted poor survival outcome in NPC patients. After adjusting for potential confounders, low expression of NKILA was confirmed to be an independent prognostic factor correlated with poor survival outcomes. Furthermore, we found that NKILA overexpression in high-metastatic-potential NPC cells repressed motile behavior and impaired the metastatic capacity in vitro and in vivo. In contrast, RNAi-mediated NKILA depletion increased the invasive motility of cells with lower metastatic potential. Further experiments demonstrated that NKILA regulated the metastasis of NPC through the NF-κB pathway. Taken together, NKILA plays vital roles in the pathogenesis of NPC. The unique histological characteristics of NPC indicate that local inflammation plays a vital role in carcinogenesis of nasopharyngeal carcinoma. [ABSTRACT FROM AUTHOR]

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA. [ABSTRACT FROM AUTHOR]

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina. [ABSTRACT FROM AUTHOR]

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world. [ABSTRACT FROM AUTHOR]

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis. [ABSTRACT FROM AUTHOR]

Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2, which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that require RAD52 vs. POLQ, and whether combined disruption of these factors causes distinct effects on genome maintenance, have been unclear. Using human U2OS cells, we generated a cell line with POLQ mutations upstream of the polymerase domain, a RAD52 knockout cell line, and a line with combined disruption of both genes. We also examined RAD52 and POLQ using RNA-interference. We find that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin, and a synthetic reduction in replication fork restart velocity. We also examined the influence of RAD52 and POLQ on several DSB repair events. We find that RAD52 is particularly important for repair using ≥ 50 nt repeat sequences that flank the DSB, and that also involve removal of non-homologous sequences flanking the repeats. In contrast, POLQ is important for repair events using 6 nt (but not ≥ 18 nt) of flanking repeats that are at the edge of the break, as well as oligonucleotide microhomology-templated (i.e., 12–20 nt) repair events requiring nascent DNA synthesis. Finally, these factors show key distinctions with BRCA2, regarding effects on DSB repair events and response to stalled replication forks. These findings indicate that RAD52 and POLQ have distinct roles in genome maintenance, including for specific features of DSB repair events, such that combined disruption of these factors may be effective for genotoxin sensitization and/or synthetic lethal strategies. [ABSTRACT FROM AUTHOR]

C-to-U editing is an important event in post-transcriptional RNA processing, which converts a specific cytidine (C)-to-uridine (U) in transcripts of mitochondria and plastids. Typically, the pentatricopeptide repeat (PPR) protein, which specifies the target C residue by binding to its upstream sequence, is involved in the editing of one or a few sites. Here we report a novel PPR-DYW protein EMP21 that is associated with editing of 81 sites in maize. EMP21 is localized in mitochondria and loss of the EMP21 function severely inhibits the embryogenesis and endosperm development in maize. From a scan of 35 mitochondrial transcripts produced by the Emp21 loss-of-function mutant, the C-to-U editing was found to be abolished at five sites (nad7-77, atp1-1292, atp8-437, nad3-275 and rps4-870), while reduced at 76 sites in 21 transcripts. In most cases, the failure to editing resulted in the translation of an incorrect residue. In consequence, the mutant became deficient with respect to the assembly and activity of mitochondrial complexes I and V. As six of the decreased editing sites in emp21 overlap with the affected editing sites in emp5-1, and the editing efficiency at rpl16-458 showed a substantial reduction in the emp21-1 emp5-4 double mutant compared with the emp21-1 and emp5-4 single mutants, we explored their interaction. A yeast two hybrid assay suggested that EMP21 does not interact with EMP5, but both EMP21 and EMP5 interact with ZmMORF8. Together, these results indicate that EMP21 is a novel PPR-DYW protein required for the editing of ~17% of mitochondrial target Cs, and the editing process may involve an interaction between EMP21 and ZmMORF8 (and probably other proteins). [ABSTRACT FROM AUTHOR]

The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. ATRIP/Ddc2 interacts with replication protein A-bound single-stranded DNA (RPA-ssDNA) and recruits ATR/Mec1 to sites of DNA damage. Mec1 is stimulated by the canonical activators including Ddc1, Dpb11 and Dna2. We have characterized the ddc2-S4 mutation and shown that Ddc2 not only recruits Mec1 to sites of DNA damage but also stimulates Mec1 kinase activity. However, the underlying mechanism of Ddc2-dependent Mec1 activation remains to be elucidated. Here we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro. The ddc2-S4 mutation diminishes damage-induced phosphorylation of the checkpoint mediators, Rad9 and Mrc1. Rad9 controls checkpoint throughout the cell-cycle whereas Mrc1 is specifically required for the S-phase checkpoint. Notably, S-phase checkpoint signaling is more defective in ddc2-S4 mutants than in cells where the Mec1 activators (Ddc1/Dpb11 and Dna2) are dysfunctional. To understand a role of Ddc2 in Mec1 activation, we reconstituted an in vitro assay using purified Mec1-Ddc2 complex, RPA and ssDNA. Whereas ssDNA stimulates kinase activity of the Mec1-Ddc2 complex, RPA does not. However, RPA can promote ssDNA-dependent Mec1 activation. Neither ssDNA nor RPA-ssDNA efficiently stimulates the Mec1-Ddc2 complex containing Ddc2-S4 mutant. Together, our data support a model in which Ddc2 promotes Mec1 activation at RPA-ssDNA tracts. [ABSTRACT FROM AUTHOR]

The placenta is the interface between maternal and fetal circulations, integrating maternal and fetal signals to selectively regulate nutrient, gas, and waste exchange, as well as secrete hormones. In turn, the placenta helps create the in utero environment and control fetal growth and development. The unique epigenetic profile of the human placenta likely reflects its early developmental separation from the fetus proper and its role in mediating maternal–fetal exchange that leaves it open to a range of exogenous exposures in the maternal circulation. In this review, we cover recent advances in DNA methylation in the context of placental function and development, as well as the interaction between the pregnancy and the environment. [ABSTRACT FROM AUTHOR]

Variation in synonymous codon usage is abundant across multiple levels of organization: between codons of an amino acid, between genes in a genome, and between genomes of different species. It is now well understood that variation in synonymous codon usage is influenced by mutational bias coupled with both natural selection for translational efficiency and genetic drift, but how these processes shape patterns of codon usage bias across entire lineages remains unexplored. To address this question, we used a rich genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina. We found that, while genome-wide relative synonymous codon usage (RSCU) for all codons was highly correlated with the GC content of the third codon position (GC3), the usage of codons for the amino acids proline, arginine, and glycine was inconsistent with the neutral expectation where mutational bias coupled with genetic drift drive codon usage. Examination between genes’ effective numbers of codons and their GC3 contents in individual genomes revealed that nearly a quarter of genes (381,174/1,683,203; 23%), as well as most genomes (308/327; 94%), significantly deviate from the neutral expectation. Finally, by evaluating the imprint of translational selection on codon usage, measured as the degree to which genes’ adaptiveness to the tRNA pool were correlated with selective pressure, we show that translational selection is widespread in budding yeast genomes (264/327; 81%). These results suggest that the contribution of translational selection and drift to patterns of synonymous codon usage across budding yeasts varies across codons, genes, and genomes; whereas drift is the primary driver of global codon usage across the subphylum, the codon bias of large numbers of genes in the majority of genomes is influenced by translational selection. [ABSTRACT FROM AUTHOR]

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology. [ABSTRACT FROM AUTHOR]

Circadian clocks control the timing of animal behavioral and physiological rhythms. Fruit flies anticipate daily environmental changes and exhibit two peaks of locomotor activity around dawn and dusk. microRNAs are small non-coding RNAs that play important roles in post-transcriptional regulation. Here we identify Drosophila miR-210 as a critical regulator of circadian rhythms. Under light-dark conditions, flies lacking miR-210 (miR-210KO) exhibit a dramatic 2 hrs phase advance of evening anticipatory behavior. However, circadian rhythms and molecular pacemaker function are intact in miR-210KO flies under constant darkness. Furthermore, we identify that miR-210 determines the evening phase of activity through repression of the cell adhesion molecule Fasciclin 2 (Fas2). Ablation of the miR-210 binding site within the 3’ UTR of Fas2 (Fas2ΔmiR-210) by CRISPR-Cas9 advances the evening phase as in miR-210KO. Indeed, miR-210 genetically interacts with Fas2. Moreover, Fas2 abundance is significantly increased in the optic lobe of miR-210KO. In addition, overexpression of Fas2 in the miR-210 expressing cells recapitulates the phase advance behavior phenotype of miR-210KO. Together, these results reveal a novel mechanism by which miR-210 regulates circadian locomotor behavior. [ABSTRACT FROM AUTHOR]

High quality reference genomes are an important resource in genomic research projects. A consequence is that DNA fragments carrying the reference allele will be more likely to map successfully, or receive higher quality scores. This reference bias can have effects on downstream population genomic analysis when heterozygous sites are falsely considered homozygous for the reference allele. In palaeogenomic studies of human populations, mapping against the human reference genome is used to identify endogenous human sequences. Ancient DNA studies usually operate with low sequencing coverages and fragmentation of DNA molecules causes a large proportion of the sequenced fragments to be shorter than 50 bp—reducing the amount of accepted mismatches, and increasing the probability of multiple matching sites in the genome. These ancient DNA specific properties are potentially exacerbating the impact of reference bias on downstream analyses, especially since most studies of ancient human populations use pseudo-haploid data, i.e. they randomly sample only one sequencing read per site. We show that reference bias is pervasive in published ancient DNA sequence data of prehistoric humans with some differences between individual genomic regions. We illustrate that the strength of reference bias is negatively correlated with fragment length. Most genomic regions we investigated show little to no mapping bias but even a small proportion of sites with bias can impact analyses of those particular loci or slightly skew genome-wide estimates. Therefore, reference bias has the potential to cause minor but significant differences in the results of downstream analyses such as population allele sharing, heterozygosity estimates and estimates of archaic ancestry. These spurious results highlight how important it is to be aware of these technical artifacts and that we need strategies to mitigate the effect. Therefore, we suggest some post-mapping filtering strategies to resolve reference bias which help to reduce its impact substantially. [ABSTRACT FROM AUTHOR]

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located 13kb to 55kb upstream of the CD36 transcriptional start site of transcript ENST00000309881 and 49kb to 92kb upstream of transcript ENST00000447544, demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 and Meg-01 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies. [ABSTRACT FROM AUTHOR]

Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes in cis. In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2, Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established. [ABSTRACT FROM AUTHOR]

5’ ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5’ monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3’ end of tRNAHis. Moreover, we were able to generate this ‘miRNA’ in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5’P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3’-fragment/’miR-4454’ levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3’-fragment processing without negatively affecting tRNAHis’s canonical function of aminoacylation. [ABSTRACT FROM AUTHOR]

For sexually reproducing organisms, production of male or female gametes depends on specifying the correct sexual identity in the germline. In D. melanogaster, Sex lethal (Sxl) is the key gene that controls sex determination in both the soma and the germline, but how it does so in the germline is unknown, other than that it is different than in the soma. We conducted an RNA expression profiling experiment to identify direct and indirect germline targets of Sxl specifically in the undifferentiated germline. We find that, in these cells, Sxl loss does not lead to a global masculinization observed at the whole-genome level. In contrast, Sxl appears to affect a discrete set of genes required in the male germline, such as Phf7. We also identify Tudor domain containing protein 5-like (Tdrd5l) as a target for Sxl regulation that is important for male germline identity. Tdrd5l is repressed by Sxl in female germ cells, but is highly expressed in male germ cells where it promotes proper male fertility and germline differentiation. Additionally, Tdrd5l localizes to cytoplasmic granules with some characteristics of RNA Processing (P-) Bodies, suggesting that it promotes male identity in the germline by regulating post-transcriptional gene expression. [ABSTRACT FROM AUTHOR]

Y chromosomes often contain amplified genes which can increase dosage of male fertility genes and counteract degeneration via gene conversion. Here we identify genes with increased copy number on both X and Y chromosomes in various species of Drosophila, a pattern that has previously been associated with sex chromosome drive involving the Slx and Sly gene families in mice. We show that recurrent X/Y co-amplification appears to be an important evolutionary force that has shaped gene content evolution of sex chromosomes in Drosophila. We also demonstrate that convergent acquisition and amplification of testis expressed gene families are common on Drosophila sex chromosomes, and especially on recently formed ones, and we carefully characterize one putative novel X/Y co-amplification system. We find that co-amplification of the S-Lap1/GAPsec gene pair on both the X and the Y chromosome occurred independently several times in members of the D. obscura group, where this normally autosomal gene pair is sex-linked due to a sex chromosome—autosome fusion. We explore several evolutionary scenarios that would explain this pattern of co-amplification. Investigation of gene expression and short RNA profiles at the S-Lap1/GAPsec system suggest that, like Slx/Sly in mice, these genes may be remnants of a cryptic sex chromosome drive system, however additional transgenic experiments will be necessary to validate this model. Regardless of whether sex chromosome drive is responsible for this co-amplification, our findings suggest that recurrent gene duplications between X and Y sex chromosomes could have a widespread effect on genomic and evolutionary patterns, including the epigenetic regulation of sex chromosomes, the distribution of sex-biased genes, and the evolution of hybrid sterility. [ABSTRACT FROM AUTHOR]

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and with limited treatment options. Recent genomic analysis of HNSCC tumors has identified several distinct molecular classes, of which the mesenchymal subtype is associated with Epithelial to Mesenchymal Transition (EMT) and shown to correlate with poor survival and drug resistance. Here, we utilize an integrated approach to characterize the molecular function of ETS1, an oncogenic transcription factor specifically enriched in Mesenchymal tumors. To identify the global ETS1 cistrome, we have performed integrated analysis of RNA-Seq, ChIP-Seq and epigenomic datasets in SCC25, a representative ETS1high mesenchymal HNSCC cell line. Our studies implicate ETS1 as crucial regulator of broader oncogenic processes and specifically Mesenchymal phenotypes, such as EMT and cellular invasion. We found that ETS1 preferentially binds cancer specific regulator elements, in particular Super-Enhancers of key EMT genes, highlighting its role as a master regulator. Finally, we show evidence that ETS1 plays a crucial role in regulating the TGF-β pathway in Mesenchymal cell lines and in leading-edge cells in primary HNSCC tumors that are endowed with partial-EMT features. Collectively our study highlights ETS1 as a key regulator of TGF-β associated EMT and reveals new avenues for sub-type specific therapeutic intervention. [ABSTRACT FROM AUTHOR]