Biology subjects

Fatma, S.

Publications and source records attributed to Fatma, S..

12 recordsLinked to original sources

bioRxiv2026

Cyclase-generated 3,5-cGMP activates PucsarTIR antiphage defense through a stepwise zig-zag aligned filament assembly

Bacteria use nucleotide second messengers to couple phage recognition to antiviral effector activation. Here, we identify Pucsar, an ofshoot of the Pycsar-related antiphage family, whose cyclases generate 3,5-cGMP from GTP, that are associated with Toll/Interleukin-1 receptor (TIR) and transmembrane (TM) effectors. In the PucsarTIR system, cGMP produced by cyclase PucC, selectively activates the cognate PucTIR NADase. X-ray crystallography shows that PucC adopts a dimeric cyclase scaffold with a guanine-compatible base-recognition pocket. Cryo-EM and mutational analyses revealed that apo-PucTIR, composed of cyclic nucleotide binding and TIR domains, is self-sequestered as an inactive tetramer, in which TIR domains are proximal but misoriented. Sequential cGMP binding remodels this assembly through partially occupied intermediates and stabilizes a fully cGMP-bound dimer, that polymerizes into a zig-zag aligned filament, creating composite NAD+-binding pockets required for efficient NADase activity. These findings establish 3',5'-cGMP as an antiphage second messenger and reveal how cyclic purine signaling converts self-sequestered TIR effectors into a NADase-competent filament.

microbiology↗

bioRxiv2026

Transposon end recognition and pairing by I-F3 CRISPR-associated transposase

To develop gene therapy tools based on CRISPR-associated transposons (CASTs), it is essential to define how transposon ends are recognized and paired during transposition. Tn7-like transposons typically contain asymmetric left- and right-end sequences that flank and define DNA cargo. However, how the transposase recognizes these different sequences and assembles them into a paired end complex for cut-and-paste transposition remains unknown. Here we present the cryo-EM structure of type I-F3 (VchCAST) CAST transposase TnsB in complex with transposon DNA ends and host factor IHF, along with biochemistry, molecular dynamics, and in vivo analyses. Our structure reveals the stoichiometry and architecture of the assembly, as well as the DNA distortions required to accommodate transposon end asymmetry. Molecular dynamics suggests that these distortions are required for the coordinated assembly of the complex. Physical pairing of asymmetric left and right ends results in a novel protein-protein interface that is required for transposition efficiency. Our findings explain how transposases regulate the pairing of transposon end sequences for high-fidelity recognition, reveal a model of transposon end synapsis, and suggest future avenues to engineer DNA cargo for genome-editing applications.

biochemistry↗

bioRxiv2026

Structure of the Type I-F3 CAST holo integration complex reveals licensing mechanisms during RNA-guided DNA integration

Precise genomic integration of large DNA cargo remains a central challenge in genome engineering. In this context, CRISPR-associated transposases (CASTs) offer a promising solution by coupling RNA-guided DNA recognition to transposon-mediated integration. Type I-F3 CASTs exhibit exceptional targeting fidelity, making them a leading focus for biotechnology and gene therapy applications. Yet, despite extensive characterization, the structural basis for RNA-guided DNA integration of type I-F3 CASTs has remained elusive. Here, using cryo-EM and functional assays, we report the structural licensing mechanism of the Vibrio cholerae CAST (VchCAST) holo integration complex (HIC). Our structures reveal that sequential conformational licensing events govern selective activation of the integration machinery at the on-target site. Different components, including Cascade-TniQ, TnsC, and TnsA/B, undergo conformational changes upon assembly into the HIC, and the catalytically competent state is stabilized only within the fully assembled complex. Finally, comparison with existing CAST integration complex structures reveals a set of conserved architectural features, suggesting a common molecular basis for RNA-guided transposition across CAST diversity.

biochemistry↗

bioRxiv2026

Programmable DNA integration with New-to-Nature tools using Computational Protein Design

Programmable integration of large DNA cargo ([≥] 2 kb), without inducing double-strand breaks, remains challenging for genome editing technologies. Current approaches have limitations in programmability, depend on co-delivery of multiple components, require multiple enzymatic steps, or have variable on-target editing outcomes. Here, we address this challenge using de novo protein design to create highly active, new-to-nature RNA-guided transposons. Our strategy exploits the modular architecture of CRISPR-associated transposons (CASTs), reconfiguring their conserved transposition machinery to interface with widely adopted Cas9. The resulting system, which we call NovoCAST, simplifies the CAST architecture from eight distinct proteins to four, establishing the simplest CAST described to date. NovoCAST exhibits sharply defined integration profiles, a 500-fold increase in activity relative to the parental PmcCAST, and general programmability. Using structural and biochemical analyses, we confirmed that the designed proteins fold and function as intended. Finally, we demonstrate robust programmable genomic integration in human cells highlighting its broad potential applications in research and therapeutics. Together, these results establish de novo protein design as a powerful strategy for engineering efficient genome-editing systems and for coupling CRISPR-mediated DNA recognition to heterologous functions through de novo designed protein interfaces.

biochemistry↗

bioRxiv2026

Aging compromises Zebrafish caudal fin regeneration by disrupting Regenerative gene networks and Cellular metabolism

Zebrafish are widely recognized as a powerful vertebrate model for studying epimorphic regeneration due to their remarkable ability to restore complex tissues. However, regenerative efficiency declines with age, potentially due to alterations in gene regulatory networks and cellular metabolism. In the present study, we investigated the molecular and bioenergetic basis of age-associated regenerative decline by comparing young adult (<1 year) and old adult (>3 years) zebrafish during caudal fin regeneration. To further examine the contribution of mitochondrial function, mitochondrial dysfunction was experimentally induced using rotenone (20 nM), a mitochondrial Complex I inhibitor. Regenerative progression was assessed morphologically at 12hpa, 1dpa, 2dpa, 3dpa, and 7dpa, revealing a pronounced delay in fin regrowth in aged and rotenone-treated fish compared with young controls. Behavioral analysis indicated subtle but non-significant changes across experimental groups. Gene expression analysis using quantitative real-time PCR revealed age- and mitochondria-associated dysregulation of key regenerative gene families involved in developmental patterning, extracellular matrix organization, cellular signaling, and mitochondrial metabolism. Proteomic profiling further identified differential expression of proteins associated with mitochondrial bioenergetics, extracellular matrix remodeling, and signaling pathways required for blastema formation and tissue outgrowth. Ultrastructural examination by transmission electron microscopy revealed pronounced mitochondrial abnormalities, including enlarged mitochondria with fragmented or disrupted cristae, in aged and rotenone-treated regenerating tissues. Collectively, our integrative analysis establishes a mechanistic link between aging, mitochondrial dysfunction, and compromised regenerative capacity in zebrafish. The findings provide broader insights into metabolic constraints underlying age-related decline in regenerative potential in vertebrates.

developmental biology↗

bioRxiv2025

Biochemical and structural characterization of the RlaP family nucleotidyltransferase potentially involved in RNA repair

Nucleotidyltransferases (NTases) of Pol{beta} superfamily are highly diverse and play many important biological functions. However, many families of NTases remain uncharacterized. Because of physical proximity of the genes encoding NTases of PF10127 family to those encoding RNA ligases involved in RNA repair, PF10127 family of NTases was named RlaP (RNA ligase-associating Pol{beta}). Here we report comprehensive characterization of two RlaP from Pseudomonas fluorescens and Pseudomonas aeruginosa (PfRlaP and PaRlaP), respectively. Our study showed that, among macromolecules isolated from E. coli cells, only RNAs are the substrates of RlaP. In vitro assays employing synthetic RNAs as substrates demonstrated that RlaP catalyzes addition of one or two nucleotide monophosphate (NMP) to the 3-hydroxyl group of RNAs, with the damaged RNAs as the preferred substrates. The crystal structure of PfRlaP provided insight into molecular recognition of RNA substrate and nucleotide triphosphate (NTP) by RlaP. Aminoacylation assays indicate that RlaP is required to restore biological function of the repaired tRNAs that have suffered excessive damage. Based on our studies, we propose an in vivo scenario where cell survival requires the involvement of RlaP in RNA repair to restore the biological function of the damaged RNAs.

biochemistry↗

bioRxiv2025

Signaling and Mechanics influence the number and size of epithelial rosettes in the migrating zebrafish Posterior Lateral Line primordium

A prepattern of Fgf signaling triggers formation of epithelial rosettes as protoneuromasts form periodically in the migrating Posterior Lateral Line primordium. However, the number and size of epithelial rosettes is influenced by the balance of mechanical interactions that promote or oppose their formation. Selective slowing of leading cells in the primordium can result in the fusion of two rosettes to form one larger one, while slowing of trailing cells can result in splitting of a previously formed rosette to form two smaller ones. These observations can be accounted for by mechanics-based models, where local interactions associated with apical constriction and cell adhesion promote formation of rosettes, while tension along the length of the primordium, influenced by the relative efficacy of leading and trailing cell migration, opposes their formation. We describe computational models that illustrate how the relative speed of leading versus trailing cells, as well as changes in cell adhesion and mechanical coupling, can influence the pattern of protoneuromast formation and deposition by the migrating primordium. Our studies illustrate how signaling and mechanics together influence morphogenesis in the migrating primordium.

developmental biology↗

bioRxiv2025

SoxB1 family members inhibit Wnt signaling to promote maturation and deposition of stable neuromasts by the zebrafish Posterior Lateral Line primordium

Periodic formation of protoneuromasts within the migrating zebrafish Posterior Lateral Line primordium serves as a model for understanding steps that determine the self-organization of organ systems in development. Protoneuromast formation is initiated by Fgf signaling at the trailing zone of the migrating primordium in response to Fgfs produced by Wnt active cells in a leading zone. Progressive restriction of an initially broad Wnt signaling domain to a smaller leading zone allows new Fgf signaling-dependent protoneuromasts to form in the wake of the shrinking Wnt system. We show Sox2 and Sox3 are expressed in nascent and maturing protoneuromasts in a trailing part of the primordium in a pattern that is complementary to Wnt activity in a leading domain, where Sox1a, is expressed. Together, these SoxB1 factors help inhibit Wnt signaling to determine effective maturation of trailing protoneuromasts and the timely deposition of stable neuromasts. Using dkk1b and atoh1b to monitor initiation and subsequent maturation of protoneuromasts, respectively, we show how Wnt signaling regulates the pace of protoneuromast maturation in the migrating primordium, and how its inhibition by SoxB1 family members ensures maturation and deposition of stable neuromasts. Together, our observations define three steps in the periodic formation of neuromasts: first, polarization of Wnt activity in the primordium; second, pattern forming step that determines periodic formation of protoneuromasts in the context of polarized Wnt activity; and third, inhibition of Wnt signaling, which is essential for stabilizing nascent neuromasts formed in the earlier pattern forming stage.

developmental biology↗

bioRxiv2025

Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation

In bacteria, spontaneous mRNAs degradation and ribotoxin-induced RNA damage are two main biological events that lead to the stall of protein translation. The ubiquitous trans-translation system as well as several alternative rescue factors (Arfs) are responsible for rescuing the stalled ribosomes caused by truncated mRNAs that lack the stop codons. To date, protein release factor homolog (PrfH) is the only factor known to rescue the stalled ribosome damaged by ribotoxins. Here we show that a subfamily of PrfH, exemplified by PrfH from Capnocytophaga gingivalis (CgPrfH), rescues both types of stalled ribosomes described above. Our in vitro biochemical assays demonstrate that CgPrfH hydrolyzes the peptides attached to P-site tRNAs when in complex with both the damaged and intact ribosomes. Two cryo-EM structures of CgPrfH in complex with the damaged and intact 70S ribosomes revealed that CgPrfH employs two different regions of the protein to recognize two different stalled ribosomes to orient the GGQ motif for peptide hydrolysis. Thus, using a combination of bioinformatic, biochemical, and structural characterization described here, we have uncovered a family of ribosome rescue factors that possesses dual activities to resolve two distinct stalled protein translation in bacteria.

biochemistry↗

bioRxiv2021

Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB

In bacteria, rescue of stalled ribosomes due to 3-truncated mRNAs is carried out by the ubiquitous trans-translation system as well as alternative ribosome-rescue factors such as ArfA and ArfB. It is unclear, however, how the stalled ribosomes caused by ribosomal damages are rescued. Here, we report that a bacterial system composed of PrfH and RtcB not only rescues a stalled ribosome resulting from a specific damage in the decoding center but also repairs the damage afterwards. Peptide release assays reveal that PrfH is only active with the damaged ribosome, but not with the intact one. A 2.55-angstrom cryo-EM structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into specific recognition of the damage site by PrfH. RNA repair assays demonstrate that PrfH-coupled RtcB efficiently repairs the damaged 30S ribosomal subunit, but not the damaged tRNAs. Thus, our studies have uncovered a biological operation by a pair of bacterial enzymes, aiming to reverse the potentially lethal damage inflicted by an invading ribotoxin for cell survival.

biochemistry↗

bioRxiv2021

Molecular mechanisms of the CdnG-Cap5 antiphage defense system employing 3',2'-cGAMP as the second messenger

Cyclic-oligonucleotide-based antiphage signaling systems (CBASS) are diverse and abundant in bacteria. Here, we present biochemical and structural characterization of two CBASS systems, composed of CdnG and Cap5, from Asticcacaulis sp. and Lactococcus lactis. We show that CdnG from Asticcacaulis sp. synthesizes 3,2-cGAMP in vitro, and 3,2-cGAMP is the biological signaling molecule that activates Cap5 for DNA degradation. Crystal structures of Cap5, together with the SAVED domain in complex with 3,2-cGAMP, provide insight into the architecture of Cap5 as well as molecular recognition of 3,2-cGAMP by the SAVED domain of Cap5. Amino acid conservation of the SAVED domain of Cap5, together with mutational studies, led us to propose a novel mechanism of Back-to-Front stacking of two SAVED domains, mediated by 3,2-cGAMP, to activate HNH nuclease domain for DNA degradation. Our study of the most abundant CBASS system provides new insight into mechanisms employed by bacteria in their conflicts against phage.

biochemistry↗

bioRxiv2020

epiGBS2: an improved protocol and automated snakemake workflow for highly multiplexed reduced representation bisulfite sequencing

epiGBS is an existing reduced representation bisulfite sequencing method to determine cytosine methylation and genetic polymorphisms de novo. Here, we present epiGBS2, an improved epiGBS laboratory protocol and user-friendly bioinformatics pipeline for a wide range of species with or without reference genome. epiGBS2 decreases costs and time investment and increases user-friendliness and reproducibility. The library protocol was adjusted to allow for a flexible choice of restriction enzymes and a double digest. Instead of fully methylated adapters, semi-methylated adapters are now used. The bioinformatics pipeline was improved in speed and integrated in the snakemake workflow management system, which now makes the pipeline easy to execute, modular, and parameter settings flexible. We also provide a detailed description of the laboratory protocol, an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.3819996), and example output.

bioinformatics↗