Drosophila RNase Z is an endonuclease. It involves in the cell growth and cell proliferation and is essential for tRNA processing. The knockdown of dRNaseZ(drosophila RNaseZ) affects not only the generation of mature tRNA but also the processing of mitochondrial mRNA. This causes growth arrest and early larval lethality (Xie et al., 2011).
Similar to Human RNaseZ, the functional mitochondrial targeting signal (MTS) and nuclear localization signal (NLS) have been identified in the drosophila dRNaseZ. The MTS of dRNaseZ is located at the N-terminus flanked by two initiating methionines. In this study, the mutant RNase Z∆MTS (RNZ∆MTS) a mutant construct lacking MTS is ubiquitously expressed in dRNaseZ knockout animal as a conditional rescue model.
Glycolysis is an energy-metabolic pathway that releases free ATP and it might compensate the energy deficiency in mitochondria. The goal of this study is to analyze the impact of dRNaseZ∆MTS on glycolysis in different developmental stages. Two genes are chosen: hexokinase(HKA) and 6-phosphofrutokinase(PFK). They are rate-limiting enzymes, requiring ATP hydrolysis. Broad is used as a developmental marker to standardize the expression levels at 3rd instar stage(Figure 1)(Andres et al., 1993).
Materials and Methods
• Fly samples: Drosophila Samples are a gift from Dr Dubrovsky’s lab.
Wild type mid 3rd instar larvae; Wild type late 3rd instar larvae; dRnaseZ∆MTS mutant larvae 14days AED(After Egg Deposition); dRnaseZ∆MTS mutant larvae 28days AED
• Whole Drosophila larvae were homogenized by TRIZOL® Reagent-Invitrogen. Total RNA was extracted and purified by using the RNeasy® Plus Mini Kit (Qiagen®, Hilden, Germany)
• Primers are designed as following list (Figure 2).
• RT-PCR was performed using the QIAGEN® One-Step RT-PCR Kit (QIAGEN®, Hilden, Germany).
• The products were seperated in the 1% agarose gel and visualized under Bio-Rad® UV trans-illuminator.
• RT-PCR products were purified using QIAquick® PCR Purification Kit
• The purified DNA products were sequenced by Genewiz Inc.(South Plainfield, NJ, USA). The sequencing results were analyzed by BLAST(Basic Local Alignment Search Tool)(BLAST, NCBI).
Metamorphosis delay with molecular evidence in the dRNaseZ∆MTS mutant
To determine which stages of drosophila dRNaseZ∆MTS mutant are, broad is used as a metamorphosis determinant. As reported previously (Figure 1. Andrew et al., 1993), broad has an increased mRNA level at late 3rd instar before pupation. The transcript of broad in the mutant is not synchronical with the wild type and delays for over two weeks. The result shown indicates that the mutant 28days AED is at the same stage with the wild type mid 3rd instar larvae (Figure 3).
The mRNA level of glycolytic gene HKA suggests that an increased glucose flux into the glycolytic pathway
As expected, by testing the transcripts of HKA and PFK genes, there is an increased mRNA level of HKA in the dRNaseZ∆MTS compared with wild type 3rd instar larvae. This data suggests that more glucose may be entering the glycolysis pathway. However, there does not appear to be any difference in the level of the PFK encoding transcript (Figure 4).
The hexokinases initiate the first step in the oxidative phosphorylation of hexoses and are viewed as the regulatory enzymes that control flux into the glycolytic pathway in response to different effectors (Hochachka et al., 1984). It has been shown that glycolytic gene expression, including hexokinase A, is increased as a response to CoVa knockdown in Drosophila S2 cells (Frejie et al., 2012). In this study, hexokinase A is chosen because of its dramatically increase in CoVa mutant in S2 cells and its distinctive intensities and patterns of selection in regulating glycolytic flux (Duvernell et al., 2000). The elevated HKA encoding transcript indicates more glucose may be entering the glycolytic pathway to produce more free ATP.
PFK is another enzyme that participates in the rate-limiting step of glycolysis. It has only isoform A in drosophila (Currie et al., 1994). No difference of mRNA level observed in this stud. One possibility is that HKA has broadened the glucose flux. Therefore, a similar amount of substrates are provided for PFK in the wild type and mutant drosophila and no more PFK is required in the dRNaseZ∆MTS mutant. Another possibility is that the activity of PFK is regulated.
How the expression of HKA and PFK is regulated in the dRnaseZ∆MTS mutant is unknown, and requires further study.
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