PLoS Genetics最新文献

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, thrives in diverse environments and holds great potential to address major challenges in medicine, agriculture, and biotechnology. As a bacterial predator it represents an alternative to traditional antimicrobial strategies to combat multidrug-resistant bacterial pathogens and prevent food waste, while the multitude of predatory enzymes it produces have potential for biotechnological applications. However, while a limited set of genetic tools exist, the lack of secretion assays and fine-tuning of secretion constrain both fundamental studies and bioengineering of B. bacteriovorus. Here, we present a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. Building on functional native and synthetic promoters from the Anderson library with varying expression levels of fluorescent reporter protein mScarletI3, we evaluated different ribosomal binding sites (RBS) to fine-tune gene expression. To examine secretion, we established a novel protocol to quantify extracellular release of a Nanoluc luciferase reporter protein in B. bacteriovorus using different native Sec-dependent signal sequences. We anticipate that the newly developed genetic toolkit and techniques will advance research on this fundamental predator-prey system, laying the foundation for its broader application and future bioengineering efforts. This work will pave the way for tailored applications of B. bacteriovorus in microbial ecology, agriculture, biotechnology, and medicine.

Background: Liver cirrhosis is a progressive chronic disease with high morbidity and mortality, thereby posing a major challenge to global health. Evidence suggests that thyroid dysfunction, particularly hypothyroidism, is linked to liver diseases. Hypothyroidism disrupts metabolism, immune homeostasis, and inflammatory pathways, processes central to cirrhosis pathophysiology. However, its causal role and molecular mechanisms remain unclear.

Methods: The study initiated by analyzing the association between thyroid dysfunction and cirrhosis through retrospective analysis of longitudinal data obtained from the Medical Information Mart for Intensive Care clinical database. To assess genetic correlation, we applied linkage disequilibrium score regression, followed by bidirectional Mendelian randomization to explore potential causal relationships. Through transcriptome-wide association studies, we identified candidate genes, which were then prioritized using a combination of weighted gene co-expression network analysis and differential gene expression data integration. To interpret the biological relevance of these genes, we conducted functional enrichment analyses. We further explored gene function at the cellular level by leveraging single-cell RNA sequencing (scRNA) to map cell-specific expression patterns, analyze intercellular communication, and simulate gene knockouts. Finally, we performed molecular docking and phenome-wide Mendelian randomization to identify potential therapeutic compounds targeting the prioritized genes.

Results: Through a combination of observational and genetic insights, we established a causal relationship between hypothyroidism and cirrhosis, identifying hypothyroidism as a risk factor for cirrhosis. Subsequent multi-omics analyses highlighted HLA-DQA1 and CD27 as potential therapeutic targets. ScRNA revealed key roles of these molecules in macrophages and CD8 ⁺ T cells, and simulated knockouts confirmed their importance in T cell activation and lymphocyte proliferation. Finally, molecular docking analysis identified glycyrrhizic acid and levothyroxine sodium as candidate drugs targeting HLA-DQA1 and CD27, while phenome-wide Mendelian randomization analysis revealed potential adverse effects associated with these targets.

Conclusions: This study is the first to reveal a causal relationship between hypothyroidism and cirrhosis, potentially driven by immune dysregulation mediated by HLA-DQA1 and CD27. These findings offer novel insights into disease progression and identify HLA-DQA1 and CD27 as potential therapeutic targets, with glycyrrhizic acid and levothyroxine sodium as promising candidate drugs.

The small GTPase CDC42 promotes axon growth through actin filament polymerization and this growth is driven by axonal localization of the mRNA encoding the prenylated CDC42 isoform (Prenyl-Cdc42). Here, we show that axonal Prenyl-Cdc42 mRNA levels and the mRNA's translation are decreased by growth-inhibiting stimulation and increased by growth-promoting stimulation. In contrast, axonal RhoA mRNA transport and translation are increased by growth-inhibiting but unaffected by growth-promoting stimuli. Localized increase in KHSRP in response to growth inhibitory stimulation, through elevation of intracellular Ca2+, promotes decrease in axonal levels of Prenyl-Cdc42 mRNA. Distinct 3'UTR motifs regulate transport and axonal levels of Prenyl-Cdc42 mRNA. KHSRP protein binds to a Prenyl-Cdc42 mRNA motif within nt 801-875 and the mRNA is remarkably increased in axons of Khsrp-/- mice. Depletion of the mRNA from sciatic nerve indicates that the increased axonal Prenyl-CDC42 contributes to the accelerated nerve regeneration when neuronal KHSRP is depleted.

Proper neural circuit organization requires individual neurons to project to their targets with high specificity. While several guidance molecules have been shown to mediate axonal fasciculation and pathfinding, less is understood about how neurons intracellularly interpret and integrate these cues. Here we provide genetic evidence that the Crk-Associated Substrate (Cas) family of intracellular adaptor proteins is required for proper fasciculation and guidance of two cortical white matter tracts: the Anterior Commissure (AC) and thalamocortical axons (TCAs). Using a Cas Triple Conditional Knock Out (Cas TcKO) mouse model, we show that Cas proteins are required for proper TCA projection by a non-neuronal cortical cell population. We also demonstrate a requirement of the β1-integrin receptor for TCA projection, similarly in a population of non-neuronal cortical cells. Additional analysis of Cas TcKO mutants reveals a role for Cas proteins in AC fasciculation, here within the neurons themselves. This AC fasciculation requirement is not phenocopied in β1-integrin deficient mutants, suggesting that Cas proteins might signal downstream of a different receptor during this axon pathfinding event. These findings implicate Cas proteins as key mediators of cortical axon tract fasciculation and guidance.

Distinguishing self from non-self is crucial to direct immune responses against pathogens. Unmodified RNAs stimulate human innate immunity, but RNA modifications suppress this response. mRNA m6A modification is essential for Arabidopsis thaliana viability. However, the molecular basis of the impact of mRNA m6A depletion is poorly understood. Here, we show that disruption of the Arabidopsis mRNA m6A writer complex triggers autoimmunity. Most gene expression changes in m6A writer complex vir-1 mutants grown at 17°C are explained by defence gene activation and are suppressed at 27°C, consistent with the frequent temperature sensitivity of Arabidopsis immunity. Accordingly, we found enhanced pathogen resistance and increased premature cell death in vir-1 mutants at 17°C but not 27°C. Global temperature-sensitive mRNA poly(A) tail length changes accompany these phenotypes. Our results demonstrate that autoimmunity is a major phenotype of mRNA m6A writer complex mutants, with important implications for interpreting the role of this modification. Furthermore, we open the broader question of whether unmodified RNA triggers immune signalling in plants.

Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. We find that amhy contributes to a higher total dosage of amh early in development and likely contributes to differential germ cell proliferation key to sex determination. The creation of sex-reversed lines also allowed us to investigate the genetic basis of secondary sex characteristics. Threespine stickleback have striking differences in behavior and morphology between sexes. Here we show one of the classic traits important for reproductive success, blue male nuptial coloration, is controlled by both sex-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the "Four Core Genotypes" model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.

Clinical studies have linked a rare form of neurological disorder to the highly conserved RNase Z gene, which encodes an endoribonuclease responsible for the processing of nuclear and mitochondrial primary tRNA transcripts. Patients harboring mutant variants of this gene exhibit a spectrum of neurological dysfunction; however, no studies to date have established the causality of RNase Z-linked neuropathology. We employed CRISPR/Cas9 technology to create flies with a neuron-specific knockout of the RNase Z gene, which is rescued with transgenes encoding a wild-type or a mutant copy of RNase Z. Neuronal activity of RNase Z is vital, as mutants display striking morphological abnormalities in central and peripheral neurons, along with attenuated motor circuit function and associative learning performance. Neuron-specific mutations of RNase Z also led to mitochondrial fragmentation and elevated ROS production. By employing the rescue transgene encoding RNase Z devoid of a mitochondrial targeting signal (MTS), we segregated the mitochondrial activity of RNase Z from that in other compartments, allowing us to assess organelle-specific contributions to disease etiology and progression. We found that mutating mitochondrial RNase Z was sufficient to induce the neuropathology in flies, as they recapitulate the salient phenotypes observed in the pan-neuronal mutants. Collectively, our study validates the pathogenicity of mutant RNase Z and establishes mitochondrial-specific contributions to neuropathology.

Circular RNAs (circRNAs) are generally considered a new class of non-coding RNA (ncRNA) that frequently appears in the eukaryotic transcriptome. In principle, circRNAs may encode proteins, as some of them are generated from exons and possess elements for internal ribosome entry. Circular RNAs have the potential to serve as an unexplored reservoir for the generation of novel proteins, yet the identification of coding-circRNAs is a daunting task. In this study, we developed a specialized strategy for the discovery of coding-circRNA by combining RNA sequencing, ribosome profiling, and mass spectrometry to find a multitude of circRNAs translated in vivo. A total of 40,084 circRNAs were found in chicken myoblasts and myotubes, and 15,332 circRNAs had a predicted open reading frame (ORF). Via ribosome footprints, we discovered that a group of circRNAs (4,069) was associated with translating ribosomes (ribo-circRNAs). Moreover, a total of 3,927 circRNAs with an infinite ORF were discovered, and 860 of them were associated with translating ribosome (ribo-no-stop-codon circRNAs). Mass spectrometry found 5 specific peptides spectra spanning a back-splice junction of circRNAs. circSIK2, one of the ribo-circRNAs, could be methylated by METTL3 and translated into SIK2-176aa, thus promoting the proliferation and differentiation of myoblasts and muscle hypertrophy. Our results suggest that many circRNAs were translating during chicken myogenesis, and METTL3 could enhance the translation of circSIK2. To the best of our knowledge, only two circRNAs translation events have been reported to be mediated by m6A. Our research would represent the third such event, and the first documented instance of a translatable circRNA in poultry.

Transfer RNAs (tRNAs) are central to protein synthesis, ensuring precise decoding of the genetic code by delivering aminoacids to the ribosome. Among all RNA species, tRNAs are the most heavily and diversely modified, with modifications playing critical roles in stability, folding, and function. Here, we present a comprehensive, isodecoder-level map of tRNA modifications in the human pathogen Vibrio cholerae. This map was generated by chemical-based sequencing methods, comparing wild-type and deletion strains. By assigning specific tRNA modifications to their cognate enzymes, we defined a comprehensive modification landscape in Vibrio cholerae and confirmed species-specific features, such as the presence of a functional TrmK enzyme, largely restricted to Gram-positive bacteria. Additionally, we detected a modification at U55 that occurs independently of TruB. To assess the biological significance of these modifications, we evaluated fitness under both standard conditions and subinhibitory antibiotic stress, and examined how modifications in the anticodon stem-loop region influence codon decoding efficiency and accuracy. Based on a comparative analysis of E. coli and V. cholerae, we discuss how species-specific differences in tRNA isodecoder gene repertoires may influence the functional impact and biological importance of tRNA modifications. This work provides the first experimentally validated, genome-wide map of tRNA modifications in V. cholerae, serving as a reference for future research into RNA modifications, translation regulation, and pathogen biology.

Ensuring diversity in genomic research is crucial to address disparities in healthcare benefits experienced by Black Americans and other minority groups. Despite progress in promoting diversity, Black Americans remain underrepresented in most genetic studies, resulting in unequal access to the benefits of genetic medicine. This study investigates trusted sources of medical and genetic testing information among Black and White Americans, identifying key factors that influence trust and participation in genetic research. Using an online survey of 1,018 participants (Black Americans n = 500, White Americans n = 518), we analyzed trust and bias ratings across various sources, including medical providers, genetic counselors, and social media. Medical providers emerged as the most trusted source for both medical and genetic information across racial groups. In terms of bias, social media was viewed as most biased and medical providers as least biased across both groups. However, Black Americans reported significantly lower trust in medical providers and scientific literature compared to White Americans. Furthermore, Black Americans expressed a stronger preference for receiving medical information from individuals of the same race or ethnicity. These findings highlight the importance of tailoring communication outlets and strategies to address the specific trust concerns of underrepresented populations. Efforts to engage Black Americans in genetic research may benefit from increased involvement of medical providers and genetic counselors, improved transparency, and culturally relevant communication. By addressing these factors, the research community can work towards reducing disparities and promoting equitable access to the benefits of genetic medicine.