RiboTag translatomic profiling of Drosophila oenocytes under aging and induced oxidative stress.

BACKGROUND: Aging is accompanied with loss of tissue homeostasis and accumulation of cellular damages. As one of the important metabolic centers, liver shows age-related dysregulation of lipid metabolism, impaired detoxification pathway, increased inflammation and oxidative stress response. However, the mechanisms for these age-related changes still remain unclear. In the fruit fly, Drosophila melanogaster, liver-like functions are controlled by two distinct tissues, fat body and oenocytes. Compared to fat body, little is known about how oenocytes age and what are their roles in aging regulation. To characterize age- and stress-regulated gene expression in oenocytes, we performed cell-type-specific ribosome profiling (RiboTag) to examine the impacts of aging and oxidative stress on oenocyte translatome in Drosophila. RESULTS: We show that aging and oxidant paraquat significantly increased the levels of reactive oxygen species (ROS) in adult oenocytes of Drosophila, and aged oenocytes exhibited reduced sensitivity to paraquat treatment. Through RiboTag sequencing, we identified 3324 and 949 differentially expressed genes in oenocytes under aging and paraquat treatment, respectively. Aging and paraquat exhibit both shared and distinct regulations on oenocyte translatome. Among all age-regulated genes, oxidative phosphorylation, ribosome, proteasome, fatty acid metabolism, and cytochrome P450 pathways were down-regulated, whereas DNA replication and immune response pathways were up-regulated. In addition, most of the peroxisomal genes were down-regulated in aged oenocytes, including genes involved in peroxisomal biogenesis factors and fatty acid beta-oxidation. Many age-related mRNA translational changes in oenocytes are similar to aged mammalian liver, such as up-regulation of innate immune response and Ras/MAPK signaling pathway and down-regulation of peroxisome and fatty acid metabolism. Furthermore, oenocytes highly expressed genes involving in liver-like processes (e.g., ketogenesis). CONCLUSIONS: Our oenocyte-specific translatome analysis identified many genes and pathways that are shared between Drosophila oenocytes and mammalian liver, highlighting the molecular and functional similarities between the two tissues. Many of these genes were altered in both oenocytes and liver during aging. Thus, our translatome analysis provide important genomic resource for future dissection of oenocyte function and its role in lipid metabolism, stress response and aging regulation.

Intestinal IRE1 Is Required for Increased Triglyceride Metabolism and Longer Lifespan under Dietary Restriction.

Dietary restriction (DR) is one of the most robust lifespan-extending interventions in animals. The beneficial effects of DR involve a metabolic adaptation toward increased triglyceride usage. The regulatory mechanism and the tissue specificity of this metabolic switch remain unclear. Here, we show that the IRE1/XBP1 endoplasmic reticulum (ER) stress signaling module mediates metabolic adaptation upon DR in flies by promoting triglyceride synthesis and accumulation in enterocytes (ECs) of the Drosophila midgut. Consistently, IRE1/XBP1 function in ECs is required for increased longevity upon DR. We further identify sugarbabe, a Gli-like zinc-finger transcription factor, as a key mediator of the IRE1/XBP1-regulated induction of de novo lipogenesis in ECs. Overexpression of sugarbabe rescues metabolic and lifespan phenotypes of IRE1 loss-of-function conditions. Our study highlights the critical role of metabolic adaptation of the intestinal epithelium for DR-induced lifespan extension and explores the IRE1/XBP1 signaling pathway regulating this adaptation and influencing lifespan.

Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.

Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans.

Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. Here, we demonstrate that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, we identify germline as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, our findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms.

hnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition.

Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition.

With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging.

Target of rapamycin (TOR) is an evolutionarily conserved nutrient-sensing protein kinase that regulates growth and metabolism in all eukaryotic cells. Studies in flies, worms, yeast, and mice support the notion that the TOR signaling network modulates aging. TOR is also emerging as a robust mediator of the protective effects of various forms of dietary restriction (DR), which can extend life span and slow the onset of certain age-related diseases across species. Here we discuss how modulating TOR signaling slows aging through downstream processes including mRNA translation, autophagy, endoplasmic reticulum (ER) stress signaling, stress responses, and metabolism. Identifying the mechanisms by which the TOR signaling network works as a pacemaker of aging is a major challenge and may help identify potential drug targets for age-related diseases, thereby facilitating healthful life span extension in humans.

A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity.

L1 is a ubiquitous interspersed repeated sequence in mammals that achieved its high copy number by autonomous retrotransposition. Individual L1 elements within a genome differ in sequence and retrotransposition activity. Retrotransposition requires two L1-encoded proteins, ORF1p and ORF2p. Chimeric elements were used to map a 15-fold difference in retrotransposition efficiency between two L1 variants from the mouse genome, T(FC) and T(Fspa), to a single amino acid substitution in ORF1p, D159H. The steady-state levels of L1 RNA and protein do not differ significantly between these two elements, yet new insertions are detected earlier and at higher frequency in T(FC), indicating that it converts expressed L1 intermediates more effectively into new insertions. The two ORF1 proteins were purified and their nucleic acid binding and chaperone activities were examined in vitro. Although the RNA and DNA oligonucleotide binding affinities of these two ORF1 proteins were largely indistinguishable, D159 was significantly more effective as a nucleic acid chaperone than H159. These findings support a requirement for ORF1p nucleic acid chaperone activity at a late step during L1 retrotransposition, extend the region of ORF1p that is known to be critical for its functional interactions with nucleic acids, and enhance understanding of nucleic acid chaperone activity.

Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1.

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons comprise a large fraction of the human and mouse genomes. The mobility of these successful elements requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. In this report, we have used limited proteolysis, filter binding, and NMR spectroscopy to characterize the global structure of ORF1p and the three-dimensional structure of a highly conserved RNA binding domain. ORF1p contains three structured regions, a coiled-coil domain, a middle domain of unknown function, and a C-terminal domain (CTD). We show that high affinity RNA binding by ORF1p requires the CTD and residues within an amino acid protease-sensitive segment that joins the CTD to the middle domain. Insights in the mechanism of RNA binding were obtained by determining the solution structure of the CTD, which is shown to adopt a novel fold consisting of a three-stranded beta sheet that is packed against three alpha-helices. An RNA binding surface on the CTD has been localized using chemical shift perturbation experiments and is proximal to residues previously shown to be essential for retrotransposition, RNA binding, and chaperone activity. A similar structure and mechanism of RNA binding is expected for all vertebrate long interspersed nuclear element-1 elements, since residues encoding the middle, protease-sensitive segment, and CTD are highly conserved.

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition.

Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3' end of ORF1, and appears to depend upon sequence rather than structure. The 40 nt intergenic region (IGR) is not essential for ORF2 IRES function or retrotransposition. Because of strong cis-preference for both proteins during L1 retrotransposition, correct stoichiometry of the two proteins can only be achieved post-transcriptionally. Although the precise stoichiometry is unknown, the retrotransposition intermediate likely contains hundreds of ORF1ps for every ORF2p, together with one L1 RNA. IRES-mediated translation initiation is a well-established mechanism of message-specific regulation, hence, unique mechanisms for the recognition and control of these two IRESes in the L1 RNA could explain differences in translational efficiency of ORF1 and ORF2. In addition, translational regulation may provide an additional layer of control on L1 retrotransposition efficiency, thereby protecting the integrity of the genome.