Open Biology

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

All cells of a multicellular organism usually share an identical genome, faithfully transmitted through successive divisions. Yet, a number of animal species deviate from this dogma, as parts of their DNA are systematically eliminated in all their somatic nuclei, in a process called Programmed DNA Elimination (PDE). PDE leads to the unexpected reorganisation of the genome at every generation in all somatic cells but its molecular mechanism, evolutionary origins, and functional significance remain unknown. This lack of understanding partially stems from limitations in genetically tractable model species. PDE can target an entire chromosome, or involve chromosome fragmentation followed by selective fragment retention and elimination, raising further questions on genome stability, genome integrity and mechanisms of DNA repair. PDE by chromosome fragmentation has been described in parasitic nematodes in the family Ascarididae, copepods in the genus Cyclops and unicellular ciliates. More recently, PDE has been discovered in three non-parasitic, lab-tractable nematode species from the Rhabditidae family, opening new perspectives. In this study, we used cytological approaches to screen 25 new Rhabditidae species for PDE. We found evidence of PDE in 17 species. Our work reveals that PDE is present in 12 out of 17 tested genera, demonstrating its widespread presence in Rhabditidae nematodes, with the notable exception of C. elegans. Genetic tools have already been established for some species. This work provides a collection of lab-tractable species that can be used to test many aspects of somatic Programmed DNA Elimination by chromosome fragmentation in animals.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented. All three structures organize as tetramers with the conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. Comparative analysis with structural neighbors from other organisms demonstrated that despite sharing <29% sequence identity, TvPPi-PFKs protomer shares overall topology with both PPi- and ATP-dependent PFKs. Mass photometry confirmed that TvPPi-PFK formed tetramers under near-physiological conditions. Unexpectedly, TvPPi-PFK crystals dephosphorylate ATP to AMP during soaking. In all three structures, either ATP or AMP is bound at the enzymes dimer interface, typical of ATP-PFKs, but a novel finding for PPi-PFKs. Furthermore, a sugar phosphate binding site was observed in proximity to the ATP-binding site. Thus, the three reported TvPPi-PFK structures validate its established PPi-dependent activity while revealing previously unreported ATP and sugar phosphate binding. This study also lays a foundation for future research into putative ATP-dependent activity of TvPPi-PFK and for evaluating known phosphofructokinase inhibitors as potential therapeutics for trichomoniasis. These findings expand our understanding of PFK superfamily diversity and support the continued exploration of TvPPi-PFK as a drug target for trichomoniasis. SynopsisThe production, crystallization, and three crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) reveal ATP binding and structural similarity to both ATP-dependent and pyrophosphate-dependent phosphofructokinases. TvPPi-PFK dephosphorylates ATP and has a novel ATP-PFK-like ATP-binding cavity.

African trypanosomes employ specialised mechanisms of membrane trafficking as a key strategy to persist in both the mammalian host and insect vector. Their survival and pathogenicity rely on the continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, the luminal proteome of the T. brucei endoplasmic reticulum (ER) and Golgi apparatus remains incompletely characterised. Here, we exploit TurboID proximity biotinylation, using the abundant ER chaperone BiP (Binding-immunoglobulin protein) as luminal bait to map the ER proteome in bloodstream and procyclic lifecycle stages of Trypanosoma brucei. Comparison with BiPN, a truncated secretory form of BiP that transits the Golgi, provides differential compartmental labelling, together identifying 366 (BiP) and 428 (BiPN) proximity partners respectively and encompassing established ER quality control machinery, secretory cargo, and Golgi proteins. Quantitative ranking of BiP labelling intensity identifies a cohort of candidate BiP interactors: the most strongly enriched is Tb927.5.1160, a protein sharing structural homology with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). Endogenous mNeonGreen tagging confirms ER localisation of TbMANF in both life cycle stages, and reciprocal manipulation of its abundance by RNAi and inducible expression produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike yeast and mammals, lacks a canonical unfolded protein response, making TbMANF the first candidate regulator of BiP activity identified in kinetoplastids. Finally, TurboID proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialised ER-continuous domain. AUTHOR SUMMARYAfrican sleeping sickness is caused by Trypanosoma brucei, a parasite that survives in the mammalian bloodstream by constantly renewing its protective protein coat. To synthesise and export this surface coat, the parasite relies on two intracellular compartments, the endoplasmic reticulum (ER) and the Golgi apparatus, which function as a quality control and sorting factory for proteins entering the secretory pathway. However, the identity of the proteins that populate these compartments in blood-stage parasites, and that maintain functioning under stress conditions, has remained poorly mapped. Here, we used an enzyme-based proximity labelling strategy that identifies neighbouring proteins in live cells without disturbing their targeting signals, generating a comprehensive protein inventory of both compartments across the two main T. brucei lifecycle stages. Among the most strongly labelled proteins was Tb927.5.1160, a protein structurally related to a mammalian regulator of the master ER chaperone BiP. Reducing or increasing the abundance of Tb927.5.1160 in parasites produced opposite changes in ER stress tolerance, identifying it as a candidate modulator of ER homeostasis in a lineage that regulates protein quality control through mechanisms distinct from those operating in yeast or human cells. Together, our findings provide a new molecular resource for understanding how T. brucei sustains secretory pathway function under the biosynthetic demands of mammalian and insect host infection.

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation. SummaryGalaviz Sarmiento et al show that the developmental regulation of kinetochore phosphorylation governs mitotic fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs during mitosis contributing to their high rate of chromosome segregation errors. While differentiation increases HEC1 phosphorylation improving chromosome segregation fidelity.

Copines are a family of calcium-dependent phospholipid-binding proteins found in most eukaryotes. The expression of multiple copine genes is dysregulated in various types of human cancers. Despite this, a common mechanistic function for copines remains unknown. We are studying copines in Dictyostelium discoideum, which has six copine genes (cpnA-cpnF). Cells lacking cpnA or cpnC (cpnA- and cpnC-, respectively) exhibit many phenotypes, including defects in development, chemotaxis, adhesion, and contractile vacuole (CV) function. To further characterize the function of copines, this study tested the hypothesis that CpnD is responsible for cellular functions distinct from CpnA and CpnC. In this study, we obtained two cpnD mutants that were generated via restriction enzyme-mediated integration (REMI) mutagenesis; one in the first exon (cpnD(i291)), and one in the second exon (cpnD(i459)) of the endogenous cpnD gene. Throughout our experiments, we found that cpnD mutants had increased cellular proliferation in both axenic and bacterial cultures. Additionally, we found that cpnD mutants exhibited precocious development and had significantly larger fruiting bodies than the parental cell line. We further investigated the morphology of cpnD mutants and found that they were significantly larger than parental cells and exhibited decreased cell-substrate adhesion. cpnD mutants also had increased activated Ras compared to the parental cell line, along with significantly smaller CVs, a phenotype that was rescued after PI3K inhibition. Finally, we found that GFP-tagged CpnD localizes to the leading edge of both randomly migrating cells and in cells responding to folate. This study is the first to describe copine proteins as having a regulatory function in Ras activation and downstream signaling effects. Additionally, this study further supports our hypothesis that copines act as nonredundant cellular proteins in Dictyostelium to regulate numerous processes.

In tunicates, larvaceans represent a fascinating case of evolution, where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the "house", a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica. We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of O. dioicas cell types in embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals.

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

FAM122A regulates cell cycle progression through inhibition of the PP2A-B55 phosphoprotein phosphatase. Recent structural work has uncovered helical elements in the N-terminus of FAM122A as binding determinants for PP2A-B55 but whether FAM122A inhibition towards PP2A-B55 is regulated is presently unclear. To address this we performed a systematic analysis of the PP2A-B55 interaction with FAM122A in cells uncovering a novel region in the C-terminus of FAM122A, spanning residues 150-170, required for binding. This C-terminal region and the N-terminal helices are both required for efficient binding to PP2A-B55 suggesting a bipartite binding mechanism. We perform amino acid resolution scans of FAM122A 150-170 uncovering several residues in this region contributing to binding including the conserved Ser158, a reported phosphorylation site. We show that Ser158 is important for PP2A-B55 inhibition in human cells as well as efficient stimulation of mitotic entry in Xenopus laevis egg extracts. In human cells and in Xenopus laevis Ser158 phosphorylation is regulated with increased occupancy correlating with cell cycle stages requiring PP2A-B55 inhibition. Collectively our work uncovers novel aspects of FAM122A interaction with PP2A-B55 and provides a possible mechanism for how the inhibitory activity of FAM122A can be regulated during the cell cycle.

The retinoblastoma protein Rb is a cell cycle inhibitor that plays a central role in regulating the G1/S cell cycle transition. Un-/hypo-phosphorylated Rb suppresses E2F transcription activity by binding to E2F/DP dimers and recruiting chromatin remodelers to prevent cells from entering S phase. For cells to progress through the G1/S transition, Rb is inactivated by two mechanisms: the "classic" pathway of Rb hyperphosphorylation by Cyclin-CDK complexes, and a recently identified "degradation" mechanism driven by the E3 ubiquitin ligase UBR5. These two pathways are interconnected, as only the un-/hypo-phosphorylated Rb can be degraded, and the hyper-phosphorylated Rb is stabilized to promote its reaccumulation in preparation for the next cell division cycle. However, the molecular basis for how Rb is stabilized upon phosphorylation remains unclear. In this study, we found that UBR5 preferentially targets chromatin-associated proteins for degradation. Since Rbs chromatin association is modulated by its phosphorylation, we hypothesized that phosphorylation may affect Rb stability by altering its chromatin association. To test this, we constructed a series of un-phosphorylatable Rb variants with graded reductions in chromatin association. Consistent with our hypothesis, we observed a strong correlation between an Rb variants chromatin association and its half-life. Fusing these Rb variants to histone H1 increased chromatin association to similar levels and equalized their protein half-lives. Taken together, these findings show how phosphorylation stabilizes Rb by promoting its dissociation from chromatin. This provides a striking example for how sub-organellar protein localization may be used to regulate stability.

Colorectal cancer across sub-Saharan Africa presents a growing global health burden, with increasing cases and mortality linked to late diagnosis, limited healthcare access and lack of effective treatments. African patients typically present with aggressive disease marked by distinct genomic signatures, indicating the need for targeted treatment approaches. We integrated genetic modelling, phenotypic scoring, imaging and biochemical analysis to explore how mutations found in individual Nigerian colorectal cancer patients influence drug responsiveness. We used the data from Cancer Genome Atlas to identify mutation profiles specific to Nigerian patients. We then generated ten stable Drosophila melanogaster personalised patient avatar lines designed to model patient genomic profiles. This study focused on three lines; each line included oncogenic RAS plus targeting patient-specific variants. These models exhibited various phenotypes including altered larval size, gut size and reduced survival. Two of the three avatar lines showed improved survival, reduced hindgut proliferation zone expansion and restored redox balance after treatment with regorafenib and trametinib. Mirroring clinical patient responses, we found that response to therapy is dependent on the specific genetic profile of the tumour. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/714433v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@110518aorg.highwire.dtl.DTLVardef@5965a0org.highwire.dtl.DTLVardef@11f16a3org.highwire.dtl.DTLVardef@744a1_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAfrican colorectal cancer showed distinct mutation patterns that contribute to tumour heterogeneity. C_LIO_LIPatient-derived Drosophila avatars were engineered using tumour-specific genetic mutations with key features of human colorectal cancer. C_LIO_LITreatment with targeted therapies showed responses patterned by tumour genotype. C_LIO_LIResponse patterns indicated the need for personalised for colorectal cancer therapies among diverse populations. C_LI

Microproteins are proteins of <100 amino acids. They represent a major, and until recently, overlooked fraction of the human proteome. However, it has now been demonstrated that many of these proteins play key roles in cellular physiology. Our group reported the expression of a microprotein expressed from an ioORF within the 53BP1 CDS arising as a result of delayed translational reinitiation mediated by a small uORF within the 5 TL. We named this microprotein SEP53BP1. We have sought to expand these studies with the ultimate aim of establishing a function for this microprotein. Although this remains elusive, we report findings providing new insights into the elements regulating its translation and demonstrate that the SEP53BP1 sequence serves as a Golgi targeting tag. Lastly, despite the fact that subunits of the proteasome feature prominently on interactome studies we were unable to demonstrate an impact of microprotein over-expression on the activities of both the proteasome and immunoproteasome.

Determining spatial and temporal gene expression is crucial for understanding animal development and physiology. Promoter reporters are powerful tools used to dissect how cis-regulatory elements and trans-acting factors control gene expression. Many fluorescent proteins used in promoter reporters, however, have long half-lives (>24 hr) which limit the study of dynamic expression. Destabilizing sequences like PEST reduce the half-life of reporter proteins to provide a more representative readout of gene expression. mlt-11 is a putative protease inhibitor known to oscillate in expression at the mRNA and protein level, yet a mlt-11p::mNeonGreen::3xFLAG::PEST::tbb-2 3UTR promoter reporter did not detectably oscillate. We systematically dissected this transgene, finding that placement of a 3xFLAG tag adjacent to a PEST sequence severely hampered oscillatory expression of a mNeonGreen promoter reporter. Surprisingly, reporter designs that effectively oscillate with a GFP or mNeonGreen fluorophore fail to oscillate when mStayGold is used, with these reporters remaining detectable over 24 hours following promoter inactivation. In addition, other tested epitopes (Myc, ALFA, OLLAS) did not hamper PEST-dependent destabilization but led to varying levels of reporter expression. This study details key considerations for designing destabilized fluorescent promoter reporters.

Unraveling how adult neurons reshape their architecture is key to understanding post-developmental plasticity. Drosophila clock neurons, which remodel their terminals on a daily basis, offer a unique model to examine the mechanisms underlying structural plasticity. In this study, we examine the impact of the experimental design on the remodeling process. We established a simple fixation protocol that preserves tissue integrity and prevents its deformation while enabling the fixation of a larger number of individuals within the appropriate time window. We show that intrinsic (i.e., targeting fluorescent reporters to the membrane) or extrinsic (i.e., temperature) variables may influence this dynamic process. Examining ex vivo preparations, we found that the s-LNv terminals display numerous thin filopodia extending from their synaptic boutons. However, these fine membrane protrusions are lost upon fixation, as they could only be accurately visualized ex vivo. Finally, we present MorphoScope, a Python-based interface that eliminates observer bias in complexity measurements. Altogether, we present a powerful and robust model to investigate the principles of adult neuronal plasticity, with implications extending beyond circadian biology.

Presynaptic homeostatic plasticity (PHP) is a potent form of homeostatic plasticity that has been documented at synapses as diverse as the glutamatergic Drosophila neuromuscular junction (NMJ), cholinergic mammalian NMJ (including human), and glutamatergic synapses in the mammalian brain. Published experimental evidence in favor of PHP in adult hippocampus and cerebellum includes patch-clamp electrophysiology, presynaptic capacitance measurement, calcium imaging, optical reporters of vesicle release and correlated three-dimensional electron microscopy. These studies are grounded in newly optimized experimental protocols that differ substantively from those typically used to study activity-dependent plasticity in neonatal and juvenile slice preparations. Here, we elaborate and extend our assays and methodologies for the study of PHP in the adult mammalian brain. Our assays are designed to optimize synapse, cell and tissue health and minimize the incorporation of unintended adverse experimental conditions that may interfere with the induction and/or expression of PHP. In addition, we provide benchmark criteria for assessment of cell health, necessary for analysis of PHP and, in so doing, advance our understanding of postsynaptic conditions necessary for PHP induction in the adult brain. Our data underscore why PHP may have been previously overlooked, inclusive of a recent manuscript challenging the robust expression of PHP in the mammalian brain (Dou et al., 2026 BioRxiv [preprint]).

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, impacting hundreds of millions of people and animals globally. Disease pathology primarily originates from host immune responses to parasite eggs, which are produced only when female schistosomes are continuously paired with males. Past research focused on pairing-dependent female sexual maturation, while scarce data exist for the males reproductive biology. In this study, we characterized the G protein-coupled receptor Smgpcr9 (Smp_244240), an orphan Class A (Rhodopsin-like) GPCR with a testis-preferential and pairing-influenced expression profile in S. mansoni males. Previous bulk RNA-seq analyses of adult worms and their isolated gonads revealed that Smgpcr9 belongs to a subgroup of GPCR genes with abundant testis-preferential and pairing-influenced transcript levels in males but low and extremely low expression in unpaired and paired females, respectively. This male-/unpaired female-biased expression pattern mirrors that of neuropeptide (npp) genes of S. mansoni such as Smnpp26 and Smnpp41. In a deorphanization approach using yeast-two-hybrid analyses, GPCR internalization experiments, bioluminescence resonance energy transfer assays, and by modeling and docking analyses, we provide first evidence that both NPPs can interact with SmGPCR9. Furthermore, we optimized a GPCR RNAi approach and achieved efficient transcript knockdown (> 90%) enabling robust functional characterization of Smgpcr9. Following RNAi, physiological and morphological analyses revealed that SmGPCR9 regulates key aspects of male reproductive biology like testis morphology and spermatogenesis. Remarkably, ovary structure and egg production were also affected in paired females post RNAi. We observed similar phenotypes plus motility constraints and reduced stem-cell proliferation in both sexes upon RNAi of Smnpp26 and Smnpp41. In all cases, RNAi downstream analyses by RT-qPCR of marker genes substantiated the observed phenotypic effects. These results strongly indicate the importance of SmGPCR9, SmNPP26, and SmNPP41 for spermatogenesis and further physiological processes in male and female S. mansoni. Author SummaryResearch of the reproductive biology of schistosomes focused mainly on females so far, which upon pairing sexually mature to produce eggs that are important for the life cycle maintenance but also for the pathogenesis of schistosomiasis, the infectious disease caused by these parasites. We investigated a yet unknown G protein-coupled receptor, Smgpcr9, which showed a testis-preferential and pairing-influenced expression profile in Schistosoma mansoni males. To this end, we optimized an RNA interference (RNAi) approach for knockdown analysis, identified neuropeptides (NPPs) as potential ligands by different biochemical approaches and modeling and docking analyses, and we investigated the roles of SmGPCR9 and two interacting NPPs, SmNPP26 and SmNPP41, by physiological, microscopical, and molecular techniques. Our results strongly suggest that SmGPCR9 and both NPPs regulate spermatogenesis. Furthermore, we detected effects on ovary morphology, egg production, and stem-cell proliferation of paired females post RNAi. Taken together, we deorphanized SmGPCR9 and showed for the first time the essential role of a so far uncharacterized GPCR and two interacting neuropeptides for spermatogenesis. Our results shed first light on spermatogenesis regulatory processes controlled by GPCRs and neuropeptides in male S. mansoni and thus expand our understanding of the roles of GPCR-NPP signaling for schistosome reproductive biology.

The biological conservation between fungi and mammals due to a common ancestor has made development of selective antifungal drugs a difficult challenge. Further complicating this situation is the selection of antifungal drug-resistant organisms during drug treatment. The pathogenic yeast Nakaseomyces glabratus (called here Candida glabrata) presents an especially challenging organism due to its tendency to frequently lose susceptibility to the major antifungal drug class the azoles. Additionally, C. glabrata develops resistance to echinocandin drugs, a second, more recently described antifungal agent at 10 times the rate of other organisms. Previous work has established that the sterol responsive transcriptional regulator Upc2A is a key determinant of azole susceptibility in C. glabrata and plays a role in echinocandin resistance. We used a biochemical approach to identify proteins that co-purified with Upc2A and identified the Ypk2 AGC kinase as an interacting protein. Strains lacking YPK2 exhibited increased susceptibility to fluconazole and the echinocandin caspofungin. A ypk2{Delta} strain failed to normally induce transcription of several ERG genes but exhibited normal induction of the CDR1 ATP-binding cassette transporter gene. Isogenic ypk2{Delta} strains were also highly susceptible to the three major classes of antifungal drugs, indicating that this kinase behaves as a multidrug susceptibility factor. RNA-seq analyses indicated that the transcriptional response to exposure is different for each drug and each response is differentially altered upon loss of Ypk2. Our data indicate that Ypk2 plays an important role in coordinating gene expression that impacts susceptibility to all major antifungal drug classes.

Museum collections of Coleoptera contain genetic material of potential interest to biotechnology, and non-destructive DNA extraction enables the preservation of important specimens with concomitant release of mitochondrial and genomic DNA. Mini-barcoding of regions of the mitochondrial cytochrome oxidase subunit I (MT-COI) gene helps identify and eliminate known species from further investigation. Here we identify a novel luciferase gene, using Consensus Degenerate Hybrid Oligonucleotide (CODEHOP) primers targeting the region of the luciferase gene spanning the fourth exon, intron, and fifth exon to detect luciferase gene content and eliminate samples containing known luciferase sequences. Biotinylated luciferase gene probes from the firefly Photinus pyralis enabled the enrichment of potential luciferase gene fragments for next-generation sequencing. A bioinformatic analysis suite was then used to identify a luciferase gene sequence from a previously unidentified firefly originally collected in Costa Rica in 2012. We demonstrate that this newly discovered luciferase, termed CRLuc, catalyses a bioluminescent reaction and we determined its emission spectra, Km for the substrates ATP and D-luciferin, and pH stability.

Background/ObjectivesRNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in human is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I could function indirectly derives from studies of its homologs in yeasts called Rpb9. Here, we endogenously humanized the rpb9 gene of the fission yeast Schizosaccharomyces pombe to examine the functional capabilities of POLR2I. MethodsWe edited the genomic rpb9 locus in S. pombe so that it encodes the human POLR2I protein, and investigated functional and structural conservation. ResultsWith our humanized yeast system, we find widespread functional complementation by human POLR2I of S. pombe rpb9 roles in yeast growth, chronological aging, and stress responses. We also find that POLR2I complements novel roles for yeast rpb9 in facultative heterochromatin assembly, resistance against the chemotherapy 5-fluorouracil, and resistance against the fungicide thiabendazole. In contrast, we find that POLR2I cannot complement the role of rpb9 in resistance against the transcription elongation inhibitor 6-azauracil (6-AU) in our system. Interestingly, POLR2I could complement 6-AU resistance if ectopically expressed. Lastly, we observe extensive structural homology between Rpb9 and POLR2I proteins. ConclusionsOur study establishes an endogenous cross-species gene complementation strategy that uncovers both conserved and rewired functions of fission yeast rpb9 and its human homolog, POLR2I. In addition to validating conserved roles, we also identified conservation of previously unrecognized roles of rpb9 in heterochromatin formation and chemoresistance.

Dual-specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) modulates the cell cycle, cell fate during development, and is deregulated in cancer and metabolic syndrome. However, only a few DYRK1B substrates have been defined, so we undertook a phosphoproteomics screen in cells that exhibit inducible DYRK1B expression. Motif analysis revealed enrichment for proline-directed serine or threonine phosphorylation sites (pSer/pThr-Pro), consistent with the consensus motif of class I DYRKs. Gene ontology analysis revealed enrichment of proteins involved in mRNA binding, mRNA processing and ribonucleoprotein complexes. Several processing body (PB) components, including DCP1A, PATL1(PAT1B), EDC3 and 4E-T, were identified as DYRK1B-inducible phosphoproteins. DYRK1B also co-immunoprecipitated with DCP1A, PAT1B, EDC3, EDC4, DDX6 and XRN1. Super-resolution microscopy demonstrated that DYRK1B co-localised with DCP1A, DCP1B and DDX6 in PBs. Activation of DYRK1B increased PB abundance, whereas inhibition, depletion or knockout of DYRK1B reduced phosphorylation of DCP1A and 4E-T and decreased PB number. Re-expression of wild type, but not kinase-dead, DYRK1B restored PB numbers in knockout cells. These findings reveal novel DYRK1B targets and establish DYRK1B as a regulator of processing body abundance. HighlightsO_LIDYRK1B induces phosphorylation of a cluster of RNA binding and processing body associated proteins. C_LIO_LIDYRK1B localises to PBs and associates with multiple PB components. C_LIO_LIDYRK1B controls P-body abundance in a kinase-dependent manner. C_LI