tRNAs serve primarily as a part of the protein synthesis machinery and play an important biological role. tRNAs are first transcribed into larger precursors with 5'- and 3'- extension. Both 5'- and 3'- end processing is crucial to yield mature functional tRNAs for aminoacylation (Hartmann et al., 2009). RNase Z gets involved in the endonucleolytic pathway of tRNA 3'end maturation. It is an endoribonuclease catalyzing specific cleavage at the 3' end of tRNA precursors (Schiffer et al., 2002). RNase Z protein has two forms: a short form of RNase ZS and a long form of RNase ZL. In the genome of Drosophila melanogaster only the long form exists (Ceballos and Vioque, 2007).
Figure 1-Developmental delay is a hallmark in the RNase Z∆MTS. A. the statistical analysis of developmental profile between wild type (WT) and mutant. Different colors present different developmental stages. Y-axis presents the percentage of individual in each developmental stage. X-axis presents days after egg deposition (AED). In 10 days AED, wild type develops from embryo to pupa to adult. However, the developmental delay occurs in the mutant primarily through the elongation at third instar larvae. At 14 days AED, mutant begins to die. At 21 days AED, only a few of them enter pupa stage. None of mutant enters adult stage and dies very quickly after pupation. B. the developmental profile between wild type and mutant.
Figure 2-Broad transcript patterns indicate developmental delay in RNase ZΔMTS Drosophila. A. RT-PCR result of broad transcripts of wild type and mutant in different developmental stages. Total RNA was isolated from wild type and mutant larvae in different stages and subjected to RT-PCR. The result was shown in a 1% agarose gel. Rp49 is used as a loading control. B. semi-quantitative results of broad transcript in each larval stage. The amount of RT-PCT products were quantified after gel electrophoresis by densitometric analysis using IMAGE J. The results were normalized to the amount of rp49. The others were compared with wild type 1d AED larvae. Results are presented as mean ± SD.
Figure 3-The transcripts of LDH indicates metabolic alteration in RNase ZΔMTS Drosophila. A. RT-PCR result of LDH transcripts of wild type and mutant in different developmental stages. B. semi-quantitative results of LDH transcript in each larval stage. Semi-quantitative analysis was performed as described in Fig.2. C. qRT-PCR result of LDH transcripts. The CT values of LDH were normalized by the CT of rp49. Results are presented as mean ± SD.
Figure 4- Anaerobic glycolysis and oxidative phophorlyation. Pyruvate usually has two metabolic pathway: converting into lactate in anaerobic glycolysis or TCA cycle in oxidative phosphorylation
Mitochondrial dRNaseZ is essential for mitochondrial tRNA processing and Drosophila development. The mutant RNase Z∆MTS(Z∆MTS) is a knockout of mitochondrial dRNaseZ generated by GAL4/UAS system. The mutant exhibits a developmental delay, especially an elongation of third instar larvae. The transcript levels of three genes that may play important roles in the elongation of third instar larvae were examined in this study. The mRNA level of broad does not increase in the mutant synchronously with wild type, but delays for about two weeks. This confirms the elongation of third instar larvae. The increased transcript of L-lactate dehydrogenase (LDH) implies a possible metabolic alteration in mutan. No significant increase at mRNA level is found in AMP-activated protein kinase (AMPK).
Key word: dRNaseZ, metamorphosis delay, metabolic alteration, broad, LDH
I would give my most sincere thank to Dr. Berish Rubin for his guidance. I would thank Xie Xie and Bo Liu, who gave me great support. I would also thank Dr. Dubrovsky’s Lab for kindly providing the Drosophila samples.
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