A Transcript Analysis Reveals the Molecular Basis for Metamorphosis Delay in RNase Z∆MTS Drosophila melanogaster




Chen Yu

Introduction

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).

Drosophila RNase ZL (dRNaseZ) is involved in both nuclear and mitochondrial tRNA 3’-end maturation. It is important in cell growth and proliferation. The mutant RNase Z∆MTS (Z∆MTS) in this study is generated by rescuing dRNaseZ knockout flies with a construct lacking mitochondrial targeting sequence (MTS).More interestingly, the mutant shows development delay, especially the elongation of third instar larvae and survives for at least 2 weeks; additionally, almost all the larvae die at the end of the third instar larvae without metamorphosis (Fig1).

The goal of this study is to analyze the developmental profile of RNase Z∆MTS at the transcript level. We hypothesize that transcript of some gene could reveal the molecular basis for metamorphosis delay in RNase Z∆MTS larvae and there may be a metabolic alteration in the mutant. Three genes are examined: broad, LDH and AMPK.

Materials and Method
Total RNA from whole Drosophila larvae was extracted and purified from homogenate using the RNeasy® Plus Mini Kit (QIAGEN).RT-PCR was performed using QIAGEN® One-Step RT-PCR Kit following the manufacturer’s instructions and the products were seperated in the 1% agarose gel. RT-PCR products were purified using QIAquick® PCR Purification Kit following the manufactures instructions. The purified products were sequenced by GENEWIZ.Real-time PCR was performed using QuantiTect® SYBR Green RT-PCR Kits following the manufactures instructions.

Results

Broad transcript patterns provide a molecular evidence for metamorphosis delay in RNase ZΔMTS Drosophila.
To examine the molecular basis for the metamorphosis delay in the mutant, the transcript of broad, a metamorphosis determinant, was studied. The mRNA level of broad increases dramatically at third instar larvae of wild type right before metamorphosis (Fig.2A). However, in the mutant, the transcript of broad does not increase synchronously with wild type but delays for over two weeks. Even in 7 days AED, the transcript of broad is still at a low level. Semi-quantitative result shows no significant differences in 1st and 2nd instar larvae but an over 7-fold enhance in wild type of 5d AED. Also, we could see the transcript level of broad increases significantly in the mutant of 21 days AED (Fig.2B). A small percentage of larvae begin to enter pupa stage just at 21 days AED.
The transcripts of LDH indicates a metabolic alteration in RNase ZΔMTS Drosophila.
To examine whether an energy-related metabolic change occurs in the mutant, the transcripts of LDH and AMPK, an energy sensory gene, were examined. We found the transcripts of LDH increase in the mutant of both second and third instar larvae. Also, mRNA level of LDH increases in the mutant of third instar larvae during the development. Unexpectedly, no significant augment is found in AMPK (Fig.3).
RT-PCR products were confirmed by sequencing and all experiments were replicated at least three times.

Disscussion

It has been reported that Drosophila broad-complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis (Felix D. Karim, 1993).The transcript patterns of broad provide the molecular evidence for the metamorphosis delay in the mutant.

A large amount of energy is required during metamorphosis. Due to the defect in mitochondria, efficiency of ATP generating is expected to decrease significantly in mitochondria. Two ATPs are generated by consuming one glucose via glycolysis pathway (Fig4). Because of the increased requirement of ATP during metamorphosis, more pyruvate is expected to generate. The mRNA of LDH increases in the mutant, which indicates a metabolic alternation in RNase ZΔMTS larvae. The increasing pattern of LDH transcript in mutant of third instar larvae seems similar to that of broad transcript in mutant of third instar larvae. It may indicate a correlation between the metamorphosis delay and metabolic alteration.

Moreover, the increased AMP/ATP ratio resulting from the mitochondrial damage may cause a higher mRNA level of AMPK, the energy sensory gene. However, no significant change of AMPK is found at transcript level. It is possible that there is an augment of Ampk at protein level or of phosphorylated Ampk, an active form, at protein activity level (Michelle L, 2010). Further study on Ampk protein level/activity is required.

Reference

Hartmann, R. K., Gossringer, M., Spath, B., Fischer, S., Marchfelder, A., 2009. The making of tRNAs and more - RNase P and tRNase Z. Prog Mol Biol Transl Sci. 85, 319-68.

Schiffer, S., Rosch, S., Marchfelder, A., 2002. Assigning a function to a conserved group of proteins: the tRNA 3'-processing enzymes. EMBO J. 21, 2769-77.

Ceballos, M., Vioque, A., 2007. tRNase Z. Protein Pept Lett. 14, 137-45.

Xie, X., Dubrovskaya, V. A., Dubrovsky, E. B., 2011. RNAi knockdown of dRNaseZ, the Drosophila homolog of ELAC2, impairs growth of mitotic and endoreplicating tissues. Insect Biochem Mol Biol.

Matthew G. Vander Heiden, Lewis C. Cantley, Craig B. Thompson. Understanding the Warburg Effect: the Metabolic Requirement of Cell Proliferation. Science 324. 1029-1033 (2009)

Michelle L. Bland, Robert J. Lee, Julie M. Magallanes, J.Kevin Foskett, Morris J. Birnbaum. AMPK supports growth in Drosophila by regulating muscle activity and nutrient uptake in the gut. Dev Bio 344. 293-303 (2010)

Felix D. Karim, Gregory M. Guild and Carl S. Thummel. The Drosophila Broad-Complex plays a key role in controlling ecdysoneregulated gene expression at the onset of metamorphosis. Development 118, 977-988 (1993)

Figures


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


Abstract
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

Full Paper

Acknowledgments

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.


This document was last modified 06/19/2012.
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