The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insights into cellular responses to impaired plastid protein homeostasis.

Germline mutation is the mechanism by which genetic variation in a population is created. Inferences derived from mutation rate models are fundamental to many population genetics methods. Previous models have demonstrated that nucleotides flanking polymorphic sites-the local sequence context-explain variation in the probability that a site is polymorphic. However, limitations to these models exist as the size of the local sequence context window expands. These include a lack of robustness to data sparsity at typical sample sizes, lack of regularization to generate parsimonious models and lack of quantified uncertainty in estimated rates to facilitate comparison between models. To address these limitations, we developed Baymer, a regularized Bayesian hierarchical tree model that captures the heterogeneous effect of sequence contexts on polymorphism probabilities. Baymer implements an adaptive Metropolis-within-Gibbs Markov Chain Monte Carlo sampling scheme to estimate the posterior distributions of sequence-context based probabilities that a site is polymorphic. We show that Baymer accurately infers polymorphism probabilities and well-calibrated posterior distributions, robustly handles data sparsity, appropriately regularizes to return parsimonious models, and scales computationally at least up to 9-mer context windows. We demonstrate application of Baymer in three ways-first, identifying differences in polymorphism probabilities between continental populations in the 1000 Genomes Phase 3 dataset, second, in a sparse data setting to examine the use of polymorphism models as a proxy for de novo mutation probabilities as a function of variant age, sequence context window size, and demographic history, and third, comparing model concordance between different great ape species. We find a shared context-dependent mutation rate architecture underlying our models, enabling a transfer-learning inspired strategy for modeling germline mutations. In summary, Baymer is an accurate polymorphism probability estimation algorithm that automatically adapts to data sparsity at different sequence context levels, thereby making efficient use of the available data.

Sulfur is an indispensable element for bacterial proliferation. Prior studies demonstrated that the human pathogen Staphylococcus aureus utilizes glutathione (GSH) as a source of nutrient sulfur; however, mechanisms of GSH acquisition are not defined. Here, we identify a five-gene locus comprising a putative ABC-transporter and predicted γ-glutamyl transpeptidase (ggt) that promotes S. aureus proliferation in medium supplemented with either reduced or oxidized GSH (GSSG) as the sole source of nutrient sulfur. Based on these phenotypes, we name this transporter operon the glutathione import system (gisABCD). Ggt is encoded within the gisBCD operon, and we show that the enzyme is capable of liberating glutamate using either GSH or GSSG as substrates, demonstrating it is a bona fide γ-glutamyl transpeptidase. We also determine that Ggt is expressed in the cytoplasm, representing only the second example of cytoplasmic Ggt localization, the other being Neisseria meningitidis. Bioinformatic analyses revealed that Staphylococcus species closely related to S. aureus encode GisABCD-Ggt homologs. However, homologous systems were not detected in Staphylococcus epidermidis. Consequently, we establish that GisABCD-Ggt provides a competitive advantage for S. aureus over S. epidermidis in a GSH- and GSSG-dependent manner. Overall, this study describes the discovery of a nutrient sulfur acquisition system in S. aureus that targets GSSG in addition to GSH and promotes competition against other staphylococci commonly associated with the human microbiota.

Predicting phenotypes from genotypes is a fundamental task in quantitative genetics. With technological advances, it is now possible to measure multiple phenotypes in large samples. Multiple phenotypes can share their genetic component; therefore, modeling these phenotypes jointly may improve prediction accuracy by leveraging effects that are shared across phenotypes. However, effects can be shared across phenotypes in a variety of ways, so computationally efficient statistical methods are needed that can accurately and flexibly capture patterns of effect sharing. Here, we describe new Bayesian multivariate, multiple regression methods that, by using flexible priors, are able to model and adapt to different patterns of effect sharing and specificity across phenotypes. Simulation results show that these new methods are fast and improve prediction accuracy compared with existing methods in a wide range of settings where effects are shared. Further, in settings where effects are not shared, our methods still perform competitively with state-of-the-art methods. In real data analyses of expression data in the Genotype Tissue Expression (GTEx) project, our methods improve prediction performance on average for all tissues, with the greatest gains in tissues where effects are strongly shared, and in the tissues with smaller sample sizes. While we use gene expression prediction to illustrate our methods, the methods are generally applicable to any multi-phenotype applications, including prediction of polygenic scores and breeding values. Thus, our methods have the potential to provide improvements across fields and organisms.

Gene expression variance has been linked to organismal function and fitness but remains a commonly neglected aspect of molecular research. As a result, we lack a comprehensive understanding of the patterns of transcriptional variance across genes, and how this variance is linked to context-specific gene regulation and gene function. Here, we use 57 large publicly available RNA-seq data sets to investigate the landscape of gene expression variance. These studies cover a wide range of tissues and allowed us to assess if there are consistently more or less variable genes across tissues and data sets and what mechanisms drive these patterns. We show that gene expression variance is broadly similar across tissues and studies, indicating that the pattern of transcriptional variance is consistent. We use this similarity to create both global and within-tissue rankings of variation, which we use to show that function, sequence variation, and gene regulatory signatures contribute to gene expression variance. Low-variance genes are associated with fundamental cell processes and have lower levels of genetic polymorphisms, have higher gene-gene connectivity, and tend to be associated with chromatin states associated with transcription. In contrast, high-variance genes are enriched for genes involved in immune response, environmentally responsive genes, immediate early genes, and are associated with higher levels of polymorphisms. These results show that the pattern of transcriptional variance is not noise. Instead, it is a consistent gene trait that seems to be functionally constrained in human populations. Furthermore, this commonly neglected aspect of molecular phenotypic variation harbors important information to understand complex traits and disease.

Global climate change is increasing both average temperatures and the frequencies of extreme high temperatures. Past studies have documented a strong negative effect of exposures to temperatures >30°C on hybrid maize yields. However, these studies could not disentangle genetic adaptation via artificial selection from changes in agronomic practices. Because most of the earliest maize hybrids are no longer available, side-by-side comparisons with modern hybrids under current field conditions are generally impossible. Here, we report on the collection and curation of 81 years of public yield trial records covering 4,730 maize hybrids, which enabled us to model genetic variation for temperature responses among maize hybrids. We show that selection may have indirectly and inconsistently contributed to the genetic adaptation of maize to moderate heat stress over this time period while preserving genetic variance for continued adaptation. However, our results reveal the existence of a genetic tradeoff for tolerance to moderate and severe heat stress, leading to a decrease in tolerance to severe heat stress over the same time period. Both trends are particularly conspicuous since the mid-1970s. Such a tradeoff poses challenges to the continued adaptation of maize to warming climates due to a projected increase in the frequency of extreme heat events. Nevertheless, given recent advances in phenomics, enviromics, and physiological modeling, our results offer a degree of optimism for the capacity of plant breeders to adapt maize to warming climates, assuming appropriate levels of R&D investment.

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain's phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.

Retrotransposons have generated about half of the human genome and LINE-1s (L1s) are the only autonomously active retrotransposons. The cell has evolved an arsenal of defense mechanisms to protect against retrotransposition with factors we are only beginning to understand. In this study, we investigate Zinc Finger CCHC-Type Containing 3 (ZCCHC3), a gag-like zinc knuckle protein recently reported to function in the innate immune response to infecting viruses. We show that ZCCHC3 also severely restricts human retrotransposons and associates with the L1 ORF1p ribonucleoprotein particle. We identify ZCCHC3 as a bona fide stress granule protein, and its association with LINE-1 is further supported by colocalization with L1 ORF1 protein in stress granules, dense cytoplasmic aggregations of proteins and RNAs that contain stalled translation pre-initiation complexes and form when the cell is under stress. Our work also draws links between ZCCHC3 and the anti-viral and retrotransposon restriction factors Mov10 RISC Complex RNA Helicase (MOV10) and Zinc Finger CCCH-Type, Antiviral 1 (ZC3HAV1, also called ZAP). Furthermore, collective evidence from subcellular localization, co-immunoprecipitation, and velocity gradient centrifugation connects ZCCHC3 with the RNA exosome, a multi-subunit ribonuclease complex capable of degrading various species of RNA molecules and that has previously been linked with retrotransposon control.

Many filamentous fungi produce plant-polysaccharide-degrading enzymes (PPDE); however, the regulatory mechanism of this process is poorly understood. A Gal4-like transcription factor, CxrA, is essential for mycelial growth and PPDE production in Penicillium oxalicum. Its N-terminal region, CxrAΔ207-733 is required for the regulatory functions of whole CxrA, and contains a DNA-binding domain (CxrAΔ1-16&Δ59-733) and a methylated arginine (R) 94. Methylation of R94 is mediated by an arginine N-methyltransferase, PRMT2 and appears to induce dimerization of CxrAΔ1-60. Overexpression of prmt2 in P. oxalicum increases PPDE production by 41.4-95.1% during growth on Avicel, compared with the background strain Δku70;hphR+. Another arginine N-methyltransferase, PRMT3, appears to assist entry of CxrA into the nucleus, and interacts with CxrAΔ1-60 in vitro under Avicel induction. Deletion of prmt3 resulted in 67.0-149.7% enhanced PPDE production by P. oxalicum. These findings provide novel insights into the regulatory mechanism of fungal PPDE production.

Finding disease-relevant tissues and cell types can facilitate the identification and investigation of functional genes and variants. In particular, cell type proportions can serve as potential disease predictive biomarkers. In this manuscript, we introduce a novel statistical framework, cell-type Wide Association Study (cWAS), that integrates genetic data with transcriptomics data to identify cell types whose genetically regulated proportions (GRPs) are disease/trait-associated. On simulated and real GWAS data, cWAS showed good statistical power with newly identified significant GRP associations in disease-associated tissues. More specifically, GRPs of endothelial and myofibroblasts in lung tissue were associated with Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease, respectively. For breast cancer, the GRP of blood CD8+ T cells was negatively associated with breast cancer (BC) risk as well as survival. Overall, cWAS is a powerful tool to reveal cell types associated with complex diseases mediated by GRPs.

Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, we aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. We found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, we propose a role for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

Adaptation of organisms to environmental change may be facilitated by the creation of new genes. New genes without homologs in other lineages are known as taxonomically-restricted orphan genes and may result from divergence or de novo formation. Previously, we have extensively characterized the evolution and origin of such orphan genes in the nematode model organism Pristionchus pacificus. Here, we employ large-scale transcriptomics to establish potential functional associations and to measure the degree of transcriptional plasticity among orphan genes. Specifically, we analyzed 24 RNA-seq samples from adult P. pacificus worms raised on 24 different monoxenic bacterial cultures. Based on coexpression analysis, we identified 28 large modules that harbor 3,727 diplogastrid-specific orphan genes and that respond dynamically to different bacteria. These coexpression modules have distinct regulatory architecture and also exhibit differential expression patterns across development suggesting a link between bacterial response networks and development. Phylostratigraphy revealed a considerably high number of family- and even species-specific orphan genes in certain coexpression modules. This suggests that new genes are not attached randomly to existing cellular networks and that integration can happen very fast. Integrative analysis of protein domains, gene expression and ortholog data facilitated the assignments of biological labels for 22 coexpression modules with one of the largest, fast-evolving module being associated with spermatogenesis. In summary, this work presents the first functional annotation for thousands of P. pacificus orphan genes and reveals insights into their integration into environmentally responsive gene networks.

Intron retention plays an important role in cancer progression and chemotherapy resistance and seems to be essential for the maintenance of genome stability in cancer. Here, our goal was to analyze the role of receptor expressed in lymphoid tissue (Relt)-like 2 (RELL2) intron 4 retention in promoting pancreatic ductal adenocarcinoma (PDAC) progression. Our results showed that intron retention (IR) occurs at the fourth intron of RELL2 transcript in gemcitabine resistant PDAC cells, however, the regulatory mechanism and the clinical implications of IR of RELL2 are unclear. Firstly, we found that RELL2 plays an anti-oncogenic role in PDAC by performing in vitro functional assays including cell proliferation, GEM cytotoxicity assay and apoptosis. Subsequently, we identified the upstream gene of RELL2, DEAH-Box Helicase 38 (DHX38), and demonstrated the direct interaction between DHX38 and RELL2 by RIP-qPCR. We also found that altered expression of DHX38 resulted in corresponding changes in intron 4 retention of RELL2. Importantly, we unveiled that overexpression of DHX38 on the basis of knocking down of the fourth intron of RELL2 resulted in an impaired intron 4 intention. Overall, our study identified a new IR site in PDAC, which could be a possible target for PDAC therapy.

[This corrects the article DOI: 10.1371/journal.pgen.1009875.].

The Gram-negative bacterial pathogen Acinetobacter baumannii is a major cause of hospital-acquired opportunistic infections. The increasing spread of pan-drug resistant strains makes A. baumannii top-ranking among the ESKAPE pathogens for which novel routes of treatment are urgently needed. Comparative genomics approaches have successfully identified genetic changes coinciding with the emergence of pathogenicity in Acinetobacter. Genes that are prevalent both in pathogenic and a-pathogenic Acinetobacter species were not considered ignoring that virulence factors may emerge by the modification of evolutionarily old and widespread proteins. Here, we increased the resolution of comparative genomics analyses to also include lineage-specific changes in protein feature architectures. Using type IVa pili (T4aP) as an example, we show that three pilus components, among them the pilus tip adhesin ComC, vary in their Pfam domain annotation within the genus Acinetobacter. In most pathogenic Acinetobacter isolates, ComC displays a von Willebrand Factor type A domain harboring a finger-like protrusion, and we provide experimental evidence that this finger conveys virulence-related functions in A. baumannii. All three genes are part of an evolutionary cassette, which has been replaced at least twice during A. baumannii diversification. The resulting strain-specific differences in T4aP layout suggests differences in the way how individual strains interact with their host. Our study underpins the hypothesis that A. baumannii uses T4aP for host infection as it was shown previously for other pathogens. It also indicates that many more functional complexes may exist whose precise functions have been adjusted by modifying individual components on the domain level.

Saccharomyces cerevisiae Pif1 is a multi-functional DNA helicase that plays diverse roles in the maintenance of the nuclear and mitochondrial genomes. Two isoforms of Pif1 are generated from a single open reading frame by the use of alternative translational start sites. The Mitochondrial Targeting Signal (MTS) of Pif1 is located between the two start sites, but a Nuclear Localization Signal (NLS) has not been identified. Here we used sequence and functional analysis to identify an NLS element. A mutant allele of PIF1 (pif1-NLSΔ) that lacks four basic amino acids (781KKRK784) in the carboxyl-terminal domain of the 859 amino acid Pif1 was expressed at wild type levels and retained wild type mitochondrial function. However, pif1-NLSΔ cells were defective in four tests for nuclear function: telomere length maintenance, Okazaki fragment processing, break-induced replication (BIR), and binding to nuclear target sites. Fusing the NLS from the simian virus 40 (SV40) T-antigen to the Pif1-NLSΔ protein reduced the nuclear defects of pif1-NLSΔ cells. Thus, four basic amino acids near the carboxyl end of Pif1 are required for the vast majority of nuclear Pif1 function. Our study also reveals phenotypic differences between the previously described loss of function pif1-m2 allele and three other pif1 mutant alleles generated in this work, which will be useful to study nuclear Pif1 functions.

While it has been appreciated for decades that prophase-arrested oocytes are transcriptionally silenced on a global level, the molecular pathways that promote silencing have remained elusive. Previous work in C. elegans has shown that both topoisomerase II (TOP-2) and condensin II collaborate with the H3K9me heterochromatin pathway to silence gene expression in the germline during L1 starvation, and that the PIE-1 protein silences the genome in the P-lineage of early embryos. Here, we show that all three of these silencing systems, TOP-2/condensin II, H3K9me, and PIE-1, are required for transcriptional repression in oocytes. We find that H3K9me3 marks increase dramatically on chromatin during silencing, and that silencing is under cell cycle control. We also find that PIE-1 localizes to the nucleolus just prior to silencing, and that nucleolar dissolution during silencing is dependent on TOP-2/condensin II. Our data identify both the molecular components and the trigger for genome silencing in oocytes and establish a link between PIE-1 nucleolar residency and its ability to repress transcription.

The successful delivery of genetic material to gametes requires tightly regulated interactions between the parental chromosomes. Central to this regulation is a conserved chromosomal interface called the synaptonemal complex (SC), which brings the parental chromosomes in close proximity along their length. While many of its components are known, the interfaces that mediate the assembly of the SC remain a mystery. Here, we survey findings from different model systems while focusing on insight gained in the nematode C. elegans. We synthesize our current understanding of the structure, dynamics, and biophysical properties of the SC and propose mechanisms for SC assembly.

Premature telomere shortening is a known factor correlated to idiopathic pulmonary fibrosis (IPF) occurrence, which is a chronic, progressive, age-related disease with high mortality. The etiology of IPF is still unknown. Here, we found that UBQLN1 plays a key role in telomere length maintenance and is potentially relevant to IPF. UBQLN1 involves in DNA replication by interacting with RPA1 and shuttling it off from the replication fork. The deficiency of UBQLN1 retains RPA1 at replication fork, hinders replication and thus causes cell cycle arrest and genome instability. Especially at telomere regions of the genome, where more endogenous replication stress exists because of G rich sequences, UBQLN1 depletion leads to rapid telomere shortening in HeLa cells. It revealed that UBQLN1 depletion also shortens telomere length at mouse lung and accelerates mouse lung fibrosis. In addition, the UBQLN1 expression level in IPF patients is downregulated and correlated to poor prognosis. Altogether, these results uncover a new role of UBQLN1 in ensuring DNA replication and maintaining telomere stability, which may shed light on IPF pathogenesis and prevention.

Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.

Human ear morphology, a complex anatomical structure represented by a multidimensional set of correlated and heritable phenotypes, has a poorly understood genetic architecture. In this study, we quantitatively assessed 136 ear morphology traits using deep learning analysis of digital face images in 14,921 individuals from five different cohorts in Europe, Asia, and Latin America. Through GWAS meta-analysis and C-GWASs, a recently introduced method to effectively combine GWASs of many traits, we identified 16 genetic loci involved in various ear phenotypes, eight of which have not been previously associated with human ear features. Our findings suggest that ear morphology shares genetic determinants with other surface ectoderm-derived traits such as facial variation, mono eyebrow, and male pattern baldness. Our results enhance the genetic understanding of human ear morphology and shed light on the shared genetic contributors of different surface ectoderm-derived phenotypes. Additionally, gene editing experiments in mice have demonstrated that knocking out the newly ear-associated gene (Intu) and a previously ear-associated gene (Tbx15) causes deviating mouse ear morphology.

The early pathogenesis and underlying molecular causes of motor neuron degeneration in Parkinson's Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to suppress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, we investigated a potential role for STING in the onset of parkin-null phenotypes. Our findings demonstrate that loss of Drosophila STING in flies rescues the thorax muscle defects and the climbing ability of parkin-/- mutants. Loss of STING also suppresses the disrupted mitochondrial morphology in parkin-/- flight muscles, suggesting unexpected feedback of STING on mitochondria integrity or activation of a compensatory mitochondrial pathway. In the animals lacking both parkin and sting, PINK1 is activated and cell death pathways are suppressed. These findings support a unique, non-canonical role for Drosophila STING in the cellular and organismal response to mitochondria stress.

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.