Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.     

AU-rich element-mediated mRNA decay can occur independently of the miRNA machinery in mouse embryonic fibroblasts and Drosophila S2-cells

AU-rich elements (AREs) are regulatory sequences located in the 3' untranslated region of many short-lived mRNAs. AREs are recognized by ARE-binding proteins and cause rapid mRNA degradation. Recent reports claimed that the function of AREs may be--at least in part--relayed through the miRNA pathway. We have revisited this hypothesis using dicer knock-out mouse embryonic fibroblasts and cultured Drosophila cells. In contrast to the published results, we find no evidence for a general requirement of the miRNA pathway in the function of AREs. Endogenous ier3 mRNA, which is known to contain a functional ARE, was degraded rapidly at indistinguishable rates in wild type and dicer knock-out mouse embryonic fibroblasts. In cultured Drosophila cells, both ARE-containing GFP reporter mRNAs and the endogenous cecA1 mRNA were resistant to depletion of the mi/siRNA factors dcr-1, dcr-2, ago1 and ago2. Furthermore, the Drosophila miRNA originally proposed to recognize AU-rich elements, miR-289, is not detectably expressed in flies or cultured S2 cells. Even our attempts to overexpress this miRNA from its genomic hairpin sequence failed. Thus, this sequence cannot serve as link between the miRNA and the AU-rich element mediated silencing pathways. Taken together, our studies in mammalian and Drosophila cells strongly argue that AREs can function independently of miRNAs.     

Dcr-1 maintains Drosophila ovarian stem cells

MicroRNAs (miRNAs) regulate gene expression by controlling the turnover, translation, or both of specific mRNAs. In Drosophila, Dicer-1 (Dcr-1) is essential for generating mature miRNAs from their corresponding precursors. Because miRNAs are known to modulate developmental events, such as cell fate determination and maintenance in many species, we investigated whether a lack of Dcr-1 would affect the maintenance of stem cells (germline stem cells, GSCs; somatic stem cells, SSCs) in the Drosophila ovary by specifically removing its function from the stem cells. Our results show that dcr-1 mutant GSCs cannot be maintained and are lost rapidly from the niche without discernable features of cell death, indicating that Dcr-1 controls GSC self-renewal but not survival. bag of marbles (bam), the gene that encodes an important differentiating factor in the Drosophila germline, however, is not upregulated in dcr-1 mutant GSCs, and its removal does not slow down dcr-1 mutant GSC loss, suggesting that Dcr-1 controls GSC self-renewal by repressing a Bam-independent differentiation pathway. Furthermore, Dcr-1 is also essential for the maintenance of SSCs in the Drosophila ovary. Our data suggest that miRNAs produced by Dcr-1 are required for maintaining two types of stem cells in the Drosophila ovary.     

Transposon Defense by Endo-siRNAs,piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2

Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.     

Small RNAs from Drosophila KC-H cells. Characterization of nuclear and cytoplasmic 7S subspecies

The metabolic characteristics and nuclear and cytoplasmic localization of Drosophila 7 S small RNAs were investigated. It was found that although most Drosophila small RNAs show electrophoretic mobilities similar to those of mammalian cells, distinct metabolic behaviour can be detected in three 7 S subspecies (d1, d3 and d4) which were found not to be class III RNAs as reported for mammalian 7 S RNAs. Drosophila d3 and d4 were found to be localized in the nucleus and to be very rapidly labelled minor subspecies turning over with a half-life of less than 6 h. Although 7 S-d1 migrates close to mammalian 7 S-K, it is a major subspecies in Drosophila, is localized mostly in the nucleus and turns over with a half-life of less than 10 h in contrast with mammalian 7 S-K which has been observed to be quite stable.     

Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes

Double-stranded RNA-binding proteins (dsRBPs), such as R2D2 and Loquacious (Loqs), function in tandem with Dicer (Dcr) enzymes in RNA interference (RNAi). In Drosophila, Dcr-1/Loqs and Dcr-2/R2D2 complexes generate microRNAs (miRNAs) and small interfering RNAs (siRNAs), respectively. Although R2D2 does not regulate siRNA production, R2D2 and Dcr-2 coordinately bind siRNAs to promote assembly of the siRNA-induced silencing (siRISC) complexes. Conversely, Loqs enhances miRNA production. It is uncertain if Dcr-1 and Loqs facilitate miRNA loading onto the miRISC complexes. Here we used loqs knockout (KO) flies to characterize the physiological functions of Loqs in the miRNA pathway. Northern analysis revealed consistent accumulation of precursor (pre)-miRNAs in loqs(KO) flies. However, the lack of Loqs had differential effects on mature miRNAs: some are diminished, whereas others maintain wild-type levels. Importantly, the data suggest that miRNA production is not the rate-limiting step of the miRNA pathway. We show that Dcr-1, but not Loqs, is critical for assembly of miRISCs by using dcr-1 or loqs null egg extract. Consistent with this, recombinant Dcr-1 could efficiently interact with miRNA duplex in the absence of Loqs. Together, our results indicate that Loqs plays a prominent role in miRNA biogenesis, but is largely dispensable for miRISC assembly. Thus, Loqs and R2D2 represent two distinct functional modes for dsRBPs in the RNAi pathways.     

Role of oxidative stress and protein oxidation in the aging process

The hypothesis is that the rate of oxygen consumption and the ensuing accrual of molecular oxidative damage constitute a fundamental mechanism governing the rate of aging is supported by several lines of evidence: (i) life spans of cold blooded animals and mammals with unstable basal metabolic rate (BMR) are extended and oxidative damage (OxD) is attenuated by an experimental decrease in metabolic rate; (ii) single gene mutations in Drosophila and Caenorhabditis elegans that extend life span almost invariably result in a generalized slowing of physiological activities, albeit via different mechanisms, affecting a decrease in OxD; (iii) caloric restriction decreases body temperature and OxD; and, (iv) results of studies on the effects of transgenic overexpressions of antioxidant enzymes are generally supportive, but quite ambiguous. It is suggested that oxidative damage to proteins plays a crucial role in aging because oxidized proteins lose catalytic function and are preferentially hydrolyzed. It is hypothesized that oxidative damage to specific proteins constitutes one of the mechanisms linking oxidative stress/damage and age-associated losses in physiological functions.     

Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor

Age-associated changes in stem cell populations have been implicated in age-related diseases, including cancer. However, little is known about the underlying molecular mechanisms that link aging to the modulation of adult stem cell populations. Drosophila midgut is an excellent model system for the study of stem cell renewal and aging. Here we describe an age-related increase in the number and activity of intestinal stem cells (ISCs) and progenitor cells in Drosophila midgut. We determined that oxidative stress, induced by paraquat treatment or loss of catalase function, mimicked the changes associated with aging in the midgut. Furthermore, we discovered an age-related increase in the expression of PVF2, a Drosophila homologue of human PDGF/VEGF, which was associated with and required for the age-related changes in midgut ISCs and progenitor cell populations. Taken together, our findings suggest that PDGF/VEGF may play a central role in age-related changes in ISCs and progenitor cell populations, which may contribute to aging and the development of cancer stem cells.     

钾离子通道蛋白Shaker对果蝇心脏衰老的保护作用

钾离子通道在心肌细胞动作电位复极过程中起着重要作用。钾离子通道蛋白种类繁多,已知钾离子通道蛋白KCNQ和HERG/eag参与心脏动作电位的形成,调节心脏收缩节律。钾离子通道蛋白Shaker是果蝇(Drosophila)体内发现的第一个电压门控钾离子通道,维持神经元和肌肉细胞的电兴奋性,但是目前其在成人心脏功能中的作用仍不清楚。本研究以果蝇为模型,高频电刺激模拟心脏应激状态,观察钾离子通道蛋白shaker基因突变体的心衰发生率。同时,利用心脏特异性启动子hand4.2Gal4特异性敲低钾离子通道蛋白Shaker的表达;果蝇成体心脏生理学功能分析系统分析了1、3、5周龄特异性敲低钾离子通道蛋白Shaker的心脏表型。结果表明,shaker基因突变将严重影响果蝇心脏抗应激能力,表现在高频电刺激后的心力衰竭发生率显著性升高;心脏特异性敲低shaker基因导致5周龄果蝇心律失常发生率显著性增加;心脏特异性敲低HDAC3将显著降低果蝇寿命。综上所述,本研究推测钾离子通道蛋白Shaker在衰老过程中维护果蝇正常的心脏功能。