Abstract Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli , the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli , generating the engineered strains K12Δ poxB and K12Δ poxB Δ pta . After 39 hours of fermentation in 500 mL shake flasks, the cytidine yields of strains K12Δ poxB and K12Δ poxB Δ pta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli .

Rapgef1 , a cell fate determinant and effector of multiple signaling events is essential for mammalian embryonic development. Here, we investigated the developmental role of rapgef1 using zebrafish as a model. We show that rapgef1 is maternally expressed and alternately spliced isoforms of its two paralogs, rapgef1a and rapgef1b show development and tissue-specific expression. CRISPR-Cas9 and morpholino-based targeting of rapgef1b resulted in developmental defects in the embryonic brain and somites. The rapgef1b morphants showed altered expression of lineage determinants of the cranial neural crest. Comparative transcriptome and altered expression analysis of morphants revealed fresh insights into the developmental functions of rapgef1b in presomitic mesoderm, and somitogenesis. During early embryonic mitoses, the morphants showed mitotic defects such as diffused spindle poles and chromosome mis-congression. Our results demonstrate that rapgef1b is required for normal embryonic mitoses, cranial neural crest specification, somitogenesis, and myogenesis during embryonic development. Significance RAPGEF1 is an important signaling factor, essential for cytoskeletal remodeling, signaling, and cell adhesion in cultured mammalian cells. The Rapgef1 knockout mice embryos fail to survive beyond implantation, strongly suggesting its essential role in embryonic development. Further, mutations in RAPGEF1 are associated with many neurological disorders like schizophrenia and intellectual disability with behavioral defects. However, the role of rapgef1 in regulating embryonic events is poorly understood. This study highlights the developmental functions of rapgef1 in lineage determination during embryonic development. We show that rapgef1b acts as an activator of canonical Wnt signaling and is essential for early neurodevelopment and somite formation. Further, rapgef1b is also required to maintain mitotic fidelity, spindle pole integrity, and chromosome congression during embryonic mitoses.

Adenosine-to-Inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes. Despite its significance, the regulatory mechanisms controlling A-to-I editing remain largely unknown. In this study, we present two complementary CRISPR-based genetic screening platforms: CREDITS ( C RISPR-based R NA EDIT ing regulator S creening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq ( s ingle- c ell C RISPR-based R NA EDIT ing seq uencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations on dozens of selected genes. Through screening 1,350 RNA-binding proteins, we identified a series of known and novel A-to-I editing regulators. Detailed mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase and ATPase activities. We demonstrate that targeting DDX39B significantly enhances the efficiency of RNA editing-based tools like CellREADR and LEAPER, and represents a promising strategy for anti-HDV therapy by modulating viral genome editing. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.

ABSTRACT Endosomal retrieval and recycling of integral cargo proteins is essential for cell, tissue and organism-level development and homeostasis and is orchestrated through a specialised retrieval sub-domain on the endosomal vacuole. However, although sub-domain dysfunction is associated with human disease our appreciation of the molecular details and functional components of the retrieval sub-domain(s) remains poorly described. Here, using comparative proximity proteomics of critical retrieval sub-domain components Retromer and Retriever, their cargo adaptors, and a component of the opposing ESCRT-degradative sub-domain, we provide a data-rich resource that identifies new molecular details of retrieval sub-domain composition and organization, including an unrecognised complexity in the interface of Retromer with RAB GTPases. Combining X-ray crystallography and in silico predictions with extensive biochemical and cellular analysis, we dissect the direct association of Retromer with RAB10 regulators DENND4A, DENND4C, TBC1D1, and TBC1D4, and the RAB35 regulator TBC1D13. Overall, we conclude that the Retromer retrieval sub-domain constitutes a major hub for the regulated switching of selected RAB GTPases and propose that this constitutes a major component of the role of Retromer in neuroprotection.

The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited eKicacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo . Using this method we examine the parasite’s pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group we determined the parasite gene known as Cp23 to be essential for survival, which was confirmed through inducible knockout in vitro and in vivo . Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is essential for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.

Enhancers play critical roles in gene expression, but a full understanding of their complex functions has yet to be defined. The cellular response to excess zinc levels in C. elegans requires the HIZR-1 transcription factor, which binds the high-zinc activation (HZA) enhancer in the promoters of multiple target genes. Cadmium hijacks the excess zinc response by binding and activating HIZR-1. By analyzing the genome-wide transcriptional response to excess zinc and cadmium, we identified two positions in the genome where head-to-head oriented genes are both induced by metals. In both examples, a single predicted HZA enhancer is positioned between the two translational start sites. We hypothesized that a single enhancer can control both head-to-head genes, an arrangement that has not been extensively characterized. To test this hypothesis, we used CRISPR genome editing to precisely delete the HZA mT enhancer positioned between mtl-2 and T08G5.1 ; in this mutant, both head-to-head genes display severely reduced zinc-activated transcription, whereas zinc-activated transcription of more distant genes was not strongly affected. Deleting the HZA cF enhancer positioned between cdr-1 and F35E8.10 caused both head-to-head genes to display reduced cadmium-activated transcription, whereas cadmium-activated transcription of more distant genes was not strongly affected. These studies rigorously document that a single HZA enhancer can control two head-to-head genes, advancing our understanding of the diverse functions of enhancers. Article Summary Enhancers are critical for gene expression, but a full understanding their functions has yet to be elucidated. We discovered two positions in the C. elegans genome where a pair of head-to-head oriented genes are both transcriptionally activated by excess zinc and/or cadmium; in both cases, one high-zinc activation (HZA) enhancer is positioned between the translation start sites. When these HZA enhancers were deleted by genome engineering, both head-to-head genes lost metal-activated transcription. These results demonstrate that a single HZA enhancer can control two head-to-head genes, advancing the understanding of enhancer function.

Abstract Plants are able to endure fluctuating environments through complex signaling networks, meticulously balancing growth decisions based on internal and external cues. Central to these networks, Sucrose non-fermenting-1 related kinase 1 (SnRK1) acts as a molecular fuel gauge that promotes survival by restraining growth and favoring catabolism under restrictive conditions. However, the detailed spatiotemporal dynamics of SnRK1's regulation of plant growth remain poorly understood given the lack of adequate tools that can capture these dynamics at cellular resolution. In this study, we employed a separation of phase-based activity reporter of kinase (SPARK)-based sensor to monitor SnRK1 activity at cellular resolution during the plant life cycle. Our findings unveiled a dual role for SnRK1: a constitutive one, tightly linked to meristematic and vascular tissues, and a dynamic one, steering growth according to energy and nutrient availability. Real-time visualization of a growing Arabidopsis root corroborated this dual role, showing SnRK1’s essential role in maintaining the apical root meristem, while also dynamically steering circadian root growth. Applying CRISPR-based tissue-specific knockout (CRISPR-TSKO) of SnRK1, confirmed SnRK1’s pivotal function in root growth and development. Our results highlight the power of ASP-SPARK for real-time, in vivo analysis of SnRK1 activity, advancing our understanding of this key metabolic regulator and paving the way for detailed insights into its relationship with plant growth and stress responses.

Abstract The proteinaceous synaptonemal complex (SC) structure forms between meiotic homologous chromosomes. Its central region (CR) consists of transverse filament and central element proteins, in Arabidopsis ZYP1 and SCEP1/SCEP2, respectively. We describe a novel CR protein in Arabidopsis. SCEP3 spatiotemporally overlaps with other CR components and is conserved in plants. In scep3 , SC formation, crossover (CO) assurance (minimum one CO per chromosome pair), CO interference (limited closely-spaced CO) and heterochiasmy (male/female CO rate difference) vanish while genome-wide and particularly female CO increase. Compared with other CR proteins, SCEP3 is also critical for some synapsis-independent CO. SCEP3 interacts with ZYP1 but loads onto recombination intermediates independent of other CR proteins. We propose SCEP3’s loading onto recombination intermediates may stabilize and/or recruit further factors such as ZYP1 to a subset of these intermediates designated to form CO. Hence, SCEP3 interlinks SC and CO formation, being structurally likely the plant ortholog of yeast Ecm11.

Abstract Genotype-informed anticancer therapies such as BRAF inhibitors can show remarkable clinical efficacy in BRAF-mutant melanoma; however, drug resistance poses a major hurdle to successful cancer treatment. Many resistance events to targeted therapies have been identified, suggesting a complex path to improve therapeutics. Here, we showed the utility of a piggyBac transposon activation mutagenesis screen for the efficient identification of genes that are resistant to BRAF inhibition in melanoma. Although several forward genetic screens performed in the same context have identified a broad range of resistance genes that poorly overlap, an integrative analysis revealed a much smaller functional diversity of resistance mechanisms, including reactivation of the MAPK pathway, PI3K-AKT pathway, and Hippo pathway, suggesting that a relatively small number of therapeutic strategies might overcome resistance manifested by a large gene set. Moreover, we illustrated the pivotal role of the Hippo pathway effector WWTR1 (TAZ ) in mediating BRAF inhibition resistance through transcriptional regulation of receptor tyrosine kinases and through interactions with the E3 ubiquitin ligase NEDD4L.

The secretion of extracellular matrix (ECM) proteins is vital to the maintenance of tissue health. One major control point of this process is the Golgi apparatus, whose dysfunction causes numerous connective tissue disorders. Golgi function is tightly linked to its structure, which is maintained by the cytoskeleton and Golgi organising proteins. We sought to investigate the role of two of these organising proteins, the golgins GMAP210 and Golgin-160, in ECM secretion. We found that loss of either protein had distinct impacts on Golgi organisation. GMAP210 loss caused cisternal fragmentation and dilation, alongside the accumulation of tubulovesicular structures. Meanwhile, Golgin-160 knockout lead to Golgi fragmentation and vesicle build-up. Nonetheless, loss of each protein had a similar impact on ECM secretion and glycosaminoglycan synthesis. We therefore propose that golgins are collectively required to create the correct physical-chemical space to support efficient ECM protein secretion and modification. This is the first time that Golgin-160 has been shown to be required for ECM secretion. Summary In this study, Thompson et al demonstrate that two cis-Golgi golgins, GMAP210 and Golgin-160, have distinct, non-redundant roles in maintaining Golgi organisation and that both are required to support the efficient secretion, assembly, and modification of extracellular matrix proteins.

ABSTRACT Vγ9Vδ2T cells have the unique ability to recognize a broad range of malignant transformed cells. The tumor targeting event involving BTN2A1 and BTN3A1 dimers on the tumor cell surface is critical, leading to full activation of the TCR. Although the molecular mechanisms governing TCR engagement and T cell activation are well-characterized, the role of Vγ9Vδ2 T cells in cancer immune surveillance remains to be fully elucidated, particularly the mechanisms that enable these cells to discriminate between healthy and malignant cells at an early stage of malignant transformation. We employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer that mimic the transformation steps leading to tumor formation. We demonstrate that various single oncogenic mutations introduced into healthy organoids or cells, are sufficient to upregulate surface expressed BTN2A1 and enable Vγ9Vδ2 TCR binding to tumor cells. However, full activation of T cells through a Vγ9Vδ2TCR required additional subsequent phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1, leading to the activating heterodimerization of BTN2A1 and 3A1. Using a protein interactome mapping pipeline, we identified PHLDB2, SYNJ2 and CARMIL1 as key players in controlling these delicate dual surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed Vγ9Vδ2TCR T cells to control tumors in vitro and in vivo, emphasizing the crucial role of these molecules from early mutagenesis, to advanced cancer stages, and highlighting the therapeutic potential of a Vγ9Vδ2TCR.

Helitrons are rolling-circle transposons that amplify through rolling-circle replication mechanism. Since Helitrons were relatively recently identified, their impact on genome evolution is still not fully understood. Here, we describe that a single Helitron subfamily specifically accumulates in the subtelomeric regions of Hydractinia symbiolongicarpus , a colonial hydrozoan cnidarian. Based on the sequence divergence, it is suggested that the Helitron subfamily underwent a burst of activity in the species’ recent history. Additionally, there is a IS3EU DNA element accumulation at the putative centromeric regions, as well as minisatellite sequences of approximately 200 bp in length extending from the telomere-side end of the Helitron towards the telomere. Phylogenetic analysis of Helitrons in the H. symbiolongicarpus genome suggests that the Helitrons underwent local propagation at the subtelomeric regions. The single Helitron subfamily, along with the consecutive minisatellite, accounts for 26.1% of the genome coverage (126 Mb of the 483 Mb genome), which collectively contribute to the genome size increase observed in H. symbiolongicarpus compared with other cnidarians. Homologous sequences of Helitron in H. symbiolongicarpus were identified in the genomes of other cnidarians, suggesting that Helitrons in hydractinia were present in at least the common ancestor of Cnidaria. Furthermore, in Nematostella vectensis , an anthozoan cnidarian, Helitrons were also accumulated at the subtelomeric regions. All these findings suggest that Helitrons constitute a common cnidarian mechanism of chromosomal extension through local amplification in subtelomeric regions, driving diverse genome expansions within the clade.