• Evidence for IRES Mediated Translation of Gurken.

      Merle, Jacob Andrew (2014)
      The gene gurken (grk) in Drosophila melanogaster is required to establish the anterior/posterior and dorsal/ventral axes of the egg. When insulin levels are high such as when food is readily available, grk is translated using a common mechanism that requires recognition of the 7-methylguanosine cap at the 5’ end of the RNA. Translation of grk requires Vasa activity which is inhibited through phosphorylation is spindle-B (spn-B) mutants. It was discovered in our lab that Insulin/Insulin-like Signaling (IIS) mutations or inhibition of TOR by rapamycin result in increased grk translation in spn-B mutants thereby overcoming this cap-dependent block. Based on these data, we hypothesize that the grk 5’ UTR contains an Internal Ribosomal Entry Site (IRES). An IRES provides an alternative mechanism of translation that occurs through use of a secondary structure in the 5’ UTR in the mRNA. The utility of the IRES is that it allows for translation of important mRNA’s even in the absence of canonical cap-dependent translation factors. These conditions can arise in nature when insulin levels or nutrient availability is low. The presence of an IRES will be explored in vivo through a comparison of transgenic reporter lines containing different luciferase reporter constructs. These constructs will be used to test the hypothesis that the grk 5’ UTR contains an IRES and examine the effect of nutrient limitation, inhibition of TOR, or IIS mutations on grk translation. To localize the IRES within the GRK 5' UTR selective deletion mutations will be made in secondary structures identified through selective 2’-Hydroxyl Acylation and Primer Extension (SHAPE) analysis. The activity of these reporter lines will be analyzed through assaying these transgenic lines. This will allow for the identification of the presence and location of the IRES within the grk 5’ UTR. Here we present data demonstrating the activity of reporter constructs that have been generated by fusion with the 5' UTR of grk. Additionally we propose a new technique using GRNA chromatography to identify the presence of an IRES as well as important secondary structures needed for IRES function using the same reporter constructs.
    • Pyruvate Kinase regulates Gurken translation by reduced TOR activity in Drosophila Melanogaster.

      Blundon, Malachi Andrew (2013-01-23)
      Gurken (Grk) expression is required to specify the polarity of the developing oocyte during Drosophila oogenesis. Proper localization and translation of grk transcripts is required to achieve proper axis specification. Gkr translation initiation has been shown to be cap-dependent and require the activity of the DEAD-box RNA helicase, Vasa. Vasa activity can be repressed by the ATR/Chk2-dependent meiotic checkpoint when DNA double strand breaks (DSBs) persist in meiosis. Unrepaired DSBs in oocyte development of spindle-class mutants activate this checkpoint and result in inefficient grk translation and loss of dorsal fates. This inefficient grk translation is thought to be related to reduced Vasa activity. In a screen for suppressors of the ventralized eggshell phenotype seen in spindle-BBU mutants, we identified a mutation in the PyK gene. We show that PyK mutations suppress the eggshell phenotype independent of the DSB repair delay and Vasa phosphorylation seen in spn-B mutants. This suggests that the eggshell phenotype is corrected by overcoming the translational block of grk transcripts seen in spindle mutants. PyK has been identified as a member of the TOR signaling pathway. Direct inhibition of the TOR kinase with rapamyacin suppresses the ventralized eggshell phenotype in spn-B mutant females. PyK modulates TOR kinase activity through the TSC1/2 heterodimer. During dietary starvation, TOR activity promotes capdependent translation by restraining the activity of the translation inhibitor eIF4E binding protein (4EBP). We hypothesize that reduced TOR activity promotes grk translation independent of the ATR/Chk2 meiotic checkpoint pathway. Recent data indicates that this may be achieved by way of IRES-dependent translation initiation of grk when TOR activity is low. This discovery suggests flies are able to maintain the translation of developmentally important transcripts such as grk during periods of nutrient limitation.