bioRxiv preprint doi: https://doi.org/10.1101/272179; this version posted February 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Ribo-tag translatomic profiling of Drosophila oenocyte reveals down-regulation of 2 peroxisome and mitochondria biogenesis under aging and oxidative stress 3 Kerui Huang1*, Wenhao Chen2, Hua Bai1* 4 5 1 Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 6 50011, USA 7 2 Department of Electrical and Computer Engineering, Iowa State University, Ames, IA 50011, 8 USA 9 10 *Corresponding Authors: 11 12 Kerui Huang 13 Phone: 515-294-3842 14 Email: [email protected] 15 16 Hua Bai 17 Phone: 515-294-9395 18 Email: [email protected] 19 20 Running title: 21 Drosophila oenocyte Ribo-tag profiling 22 23 24 25 26 27 28 29 30 31 bioRxiv preprint doi: https://doi.org/10.1101/272179; this version posted February 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 Abstract 33 Background: Reactive oxygen species (ROS) has been well established as toxic, owing to its 34 direct damage on genetic materials, protein and lipids. ROS is not only tightly associated with 35 chronic inflammation and tissue aging but also regulates cell proliferation and cell signaling. Yet 36 critical questions remain in the field, such as detailed genome-environment interaction on aging 37 regulation, and how ROS and chronic inflammation alter genome that leads to aging. Here we 38 used cell-type-specific ribosome profiling (Ribo-tag) to study the impacts of aging and oxidative 39 stress on the expression of actively translated mRNA in Drosophila oenocytes, a specialized 40 hepatocyte-like cells and an understudied insect tissue. 41 Results: Through ribosome profiling, we obtain oenocyte translatome, from which we identify 42 genes specifically expressed in adult oenocyte. Many of them are cuticular proteins, including 43 tweedle family proteins, which are critical for structural constituent of cuticle. Oenocytes are 44 enriched with genes involved in unsaturated fatty acids, ketone body synthesis and degradation, 45 and xenobiotics. Aging and paraquat exhibit distinct regulation on oenocyte translatome. 3411 46 genes are differentially regulated under aging, only 1053 genes under paraquat treatment. Gene 47 set enrichment analysis identify pathways with overall declined expression under aging and 48 paraquat treatment, especially peroxisome, mitochondrial function and metabolic pathways. In 49 comparing to human liver-elevated proteins, we identify orthologues specifically enriched in 50 oenocyte. Those orthologues are involved in liver ketogenesis, ethanol metabolism, and glucose 51 metabolism. Gene Ontology (GO) analysis shows different functions between oenocyte and fat 52 body, which is another widely accepted model for liver in Drosophila. Lastly, 996 genes are 53 regulated to paraquat stress in young age, however in aged flies, the number decreased to 385. 54 Different sets of genes are regulated in response to stress between young and old flies. 55 Conclusions: Our analysis reveals 17 oenocyte specific genes, which help to define oenocyte’s 56 function. Oenocyte shares some aspects of human liver function, makes the oenocytes applicable 57 as a model system. Our gene set analysis provides insight into fundamental changes of oenocyte 58 under aging and oxidative stress. The finding that peroxisome is down-regulated under oxidative 59 stress suggests peroxisome plays important role in regulating oenocyte homeostasis under aging, 60 which can have a significant impact on whole body and fat metabolism. 61 Keywords: oxidative stress, aging, oenocyte, hepatocyte-like cells, ribosomal profiling, 62 mitochondria ribosomal proteins, peroxisome bioRxiv preprint doi: https://doi.org/10.1101/272179; this version posted February 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 63 Introduction 64 Oenocyte 65 Oenocytes are secretory cells that are found in most pterygote insects [1]. Oenocyte is 66 important for growth, because oenocyte ablation will trigger growth arrest and lethality before 67 reaching pupariation [2]. Several studies have attempted to decipher oenocyte’s function on 68 transcriptional level. Studies have shown that 51 transcripts are enriched in larvae oenocytes, and 69 22 of them have human homologues that are fat-metabolizing genes expressed in hepatocytes 70 [2]. Chatterjee et al., 2014 conducted tissue-specific expression profiling on oenocyte, 71 confirming its role on lipid mobilization during fasting. Transcriptome of oenocytes from Aedes 72 aegypti pupae reveals that oenocyte highly expresses transcripts from cytochrome P450 73 superfamily, whose members are crucial for detoxification and lipid metabolism [3]. Despite the 74 importance of oenocyte, a comprehensive understanding on Drosophila adult oenocyte function 75 is still lacking, mainly due to the challenge for obtaining samples without contamination. 76 Hall marks of aging and Oxidative stress 77 Aging has been characterized as gradual decline of cell physiological integrity and 78 maintenance, leading to cell death and vulnerability to diseases. The topic has embarked 79 extensive research and investigation. Recently molecular and cellular hallmarks of aging have 80 been identified. The nine hallmarks are considered to contribute to aging process and lead to the 81 aging phenotypes. Amelioration of these processes can retard normal aging process and increase 82 lifespan. The nine hallmarks are: 1) genomic instability, because of DNA damaged by 83 endogenous and exogenous threats, including reactive oxygen species (ROS). 2) Telomere 84 attribution. 3) Epigenetic alterations involve modification of histones, DNA methylation and 85 chromatin remodeling. 4) Loss of proteostasis involves inability to stabilize folded proteins 86 through chaperone, and failure of protein degradation by lysosome or proteasome. 5) 87 Dysregulated nutrient sensing, including insulin signaling pathway, nutrient-sensing systems. 6) 88 Mitochondrial dysfunction with aging results in increased production of ROS, which can cause 89 further mitochondrial damage and cellular damage. 7) Cellular senescence. 8) stem cell 90 exhaustion. 9) Altered intercellular communication [4]. 91 Liver aging 92 Aging is the major risk factor for chronic liver diseases. Studies have found that aging is 93 associated with alterations of hepatic structure, function as well as changes in liver cells. For bioRxiv preprint doi: https://doi.org/10.1101/272179; this version posted February 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 94 example, the volume and blood flow of liver decreases with aging, diminished regenerative 95 ability, as well as declines in the Phase I metabolism of certain drugs. The blood cholesterol, 96 neutral fat levels and high-density lipoprotein cholesterol increases with time, which will 97 eventually lead to non-alcoholic fatty liver disease (NAFLD). The metabolism for low-density 98 lipoprotein cholesterol decreases by 35% [5]. Aging leads to be polyploidy nuclei, decreased 99 area of smooth endoplasmic reticulum, which reduce the synthesis of microsomal proteins in the 100 liver and dysfunction of mitochondria [6]. Aging alters stress-induced expression of heme 101 oxygenase-1, which may lead to lower stress tolerance and sensitivity [7]. In addition, a recent 102 study indicated that age-associated change on CCAAT/enhancer-binding protein (C/EBP) 103 protein family caused liver injury after carbon tetrachloride (CCl4) treatments [8]. They observed 104 altered chromatin structure in old wild type mice and young mice which express an aged-like 105 isoform of C/EBPɑ. They also found age-related repression on C/EBPɑ, Farnesoid X Receptor 106 (FXR) and telomere reverse transcriptase [9]. Molecular mechanism for altered liver function 107 during aging include increased ROS formation, DNA damage, activation of p300-C/EBP- 108 dependent neutral fat synthesis [10], a decreased autophagy [11], increased M1 macrophage 109 inflammatory responses [12], and activation of nuclear factor-휅B pathways [13,14]. Despite of 110 many advances in detailed genetic control of aging, very little is known about how translatome 111 changes with liver aging and what initiated the aging process. 112 Oxidative stress generated by imbalanced ROS level could be the key cause for liver 113 damage during aging. Aging is accompanied by increased level of ROS [15], which can damage 114 cellular contents. Paradoxically elevated ROS can act as signaling molecules in maintaining 115 cellular function, a process termed redox biology. Evidence suggests that increasing ROS can 116 promote healthy aging by activating stress-response pathways [16]. The detailed molecular 117 mechanism for redox biology induced pathology or beneficial effect is pressingly needed to 118 solve this paradox. Moreover, what cellular functions changed during liver aging are caused by 119 redox biology is still unknown. 120 The aim of this study is to establish Drosophila oenocyte as a liver model to study its 121 translatome changes