Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sensitivity to DNA damaging reagents were identified using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway. Copyright (C) 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Microglia are tissue-resident macrophages residing in the central nervous system (CNS) and play critical roles in removing cellular debris and infectious agents as well as regulating neurogenesis and neuronal activities. Yet, the molecular basis underlying the establishment of microglia pool and the maintenance of their homeostasis in the CNS remain largely undefined. Here we report the identification and characterization of a mutant zebrafish, which harbors a point mutation in the nucleotide-binding oligomerization domain (NOD) like receptor gene nlrc3-like, resulting in the loss of microglia in a temperature sensitive manner. Temperature shift assay reveals that the late onset of nlrc3-like deficiency leads to excessive microglia cell death. Further analysis shows that the excessive microglia death in nlrc3-like deficient mutants is attributed, at least in part, to aberrant activation of canonical inflammasome pathway. Our study indicates that proper regulation of inflammasome cascade is critical for the maintenance of microglia homeostasis. Copyright (C) 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans. Here, we identified a novel Drosophila protein radial spoke binding protein 15 (RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles (SVs), asynchronous individualization complex (IC) and defective "9 + 2" structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD_R_PIKA superfamily domain which is important for the stabilization of dRSPH3. Moreover, loss of dRSPH3, as well as dRSPH1, dRSPH4a and dRSPH9, showed similar phenotypes to rsbp15(KO) mutant. Together, our results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella. Copyright (C) 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Grain weight and grain number are two important traits directly determining grain yield in rice. To date, a lot of genes related to grain weight and grain number have been identified; however, the regulatory mechanism underlying these genes remains largely unknown. In this study, we studied the biological function of OsSPL18 during grain and panicle development in rice. Knockout (KO) mutants of OsSPL18 exhibited reduced grain width and thickness, panicle length and grain number, but increased tiller number. Cytological analysis showed that OsSPL18 regulates the development of spikelet hulls by affecting cell proliferation. qRT-PCR and GUS staining analyses showed that OsSPL18 was highly expressed in developing young panicles and young spikelet hulls, in agreement with its function in regulating grain and panicle development. Transcriptional activation experiments indicated that OsSPL18 is a functional transcription factor with activation domains in both the N-terminus and C-terminus, and both activation domains are indispensable for its biological functions. Quantitative expression analysis showed that DEP1, a major grain number regulator, was significantly down-regulated in OsSPL18 KO lines. Both yeast one-hybrid and dual-luciferase (LUC) assays showed that OsSPL18 could bind to the DEP1 promoter, suggesting that OsSPL18 regulates panicle development by positively regulating the expression of DEP1. Sequence analysis showed that OsSPL18 contains the OsmiR156k complementary sequence in the third exon; 5' RLM-RACE experiments indicated that OsSPL18 could be cleaved by OsmiR156k. Taken together, our results uncovered a new OsmiR156k-OsSPL18-DEP1 pathway regulating grain number in rice. Copyright (C) 2019, The Authors. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press.
Brain-derived neurotrophic factor (BDNF) plays a crucial role in human obesity. Yet, the neural circuitry supporting the BDNF-mediated control of energy homeostasis remains largely undefined. To map key regions that might provide inputs to or receive inputs from the paraventricular nucleus (PVN) BDNF neurons, a key type of cells in regulating feeding and thermogenesis, we used rabies virus-based transsynaptic labeling and adeno-associated virus based anterograde tracing techniques to reveal their whole-brain distributions. We found that dozens of brain regions provide dense inputs to or receive dense inputs from PVN BDNF neurons, including several known weight control regions and several novel regions that might be functionally important for the BDNF-mediated regulation of energy homeostasis. Interestingly, several regions show very dense reciprocal connections with PVN BDNF neurons, including the lateral septum, the preoptic area, the ventromedial hypothalamic nucleus, the paraventricular thalamic nucleus, the zona incerta, the lateral parabrachial nucleus, the subiculum, the raphe magnus nucleus, and the raphe pallidus nucleus. These strong anatomical connections might be indicative of important functional connections. Therefore, we provide an outline of potential neural circuitry mediated by PVN BDNF neurons, which might be helpful to resolve the complex obesity network. Copyright (C) 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled. Copyright (C) 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Mutations in long-chain acyl-CoA synthetase 4 (ACSL4) are associated with non-syndromic X-linked intellectual disability (ID). However, the neural functions of ACSL4 and how loss of ACSL4 leads to ID remain largely unexplored. We report here that mutations in Acsl, the Drosophila ortholog of human ACSL3 and ACSL4, result in developmental defects of the mushroom body (MB), the center of olfactory learning and memory. Specifically, Acsl mutants show fewer MB neuroblasts (Nbs) due to reduced proliferation activity and premature differentiation. Consistently, these surviving Nbs show reduced expression of cyclin E, a key regulator of the G1- to S-phase cell cycle transition, and nuclear mislocalization of the transcriptional factor Prospero, which is known to repress self-renewal genes and activate differentiating genes. Furthermore, RNA-seq analysis reveals downregulated Nb- and cell-cycle-related genes and upregulated neuronal differentiation genes in Acsl mutant Nbs. As Drosophila Acsl and human ACSL4 are functionally conserved, our findings provide novel insights into a critical and previously unappreciated role of Acsl in neurogenesis and the pathogenesis of ACSL4-related ID. Copyright (C) 2018, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
The ability to fix heterosis or hybrid vigor in crops without crossing holds great potential for crop yield improvement and global food security. Recent studies in Nature and Nature Biotechnology from two independent groups demonstrate the feasibility of clonal propagation of hybrids by engineering changes in meiosis and embryogenesis. These synthetic apomixis technologies represent a watershed moment for both understanding the fundamental basis of heterosis and plant breeding.