Retrograde signaling targets under conditions of mitochondria failure




Ekaterina Migunova

Introduction

Eucaryotic cells have two different sets of tRNAs which are encoded by nuclear and mitochondrial DNA.Both populations of tRNAs go through maturation process. One of the enzymes essential for this process is RNaseZ endonuclease. In mitochondria endonucleolytic activity of RNAseZ serves not only to edit tRNAs but also is essential for processing of mRNAs coding for subunits of mitochondrial respiratory chain.
When in transgenic flies Z∆MTS RNaseZ is removed from mitochondria, the function of mitochondrial respiratory chain fails. Interestingly a dramatic increase in amount of mitochondrial DNA and mass is observed in such cells (figure 2). The mitochondrial DNA replication process is controlled by nucleus, therefore the goal of this study is to identify the genes which are targeted by retrograde signalling from mitochondria in order to increase mitochondrial biogenesis.


Materials and methods


- Drosophila melanogaster flies were kindly provided by Dr. Dubrovsky’s lab. Transgenic line Z∆MTS was created to exclude RNaseZ from mitochondria
- Imaginal discs were dissected from mutant and wild type larvae of appropriate age.Wild type late 3rd instar larvae 5 AED; Z∆MTS mutant larvae 7 AED(After Egg Deposition)
- RNA was extracted using a QIAGEN RNeasy Plus Mini Kit
- RT-PCR was done with 3 different pairs of primers for each gene using One-Step RT-PCR Kit (QIAGEN®)
- The products were separated and visualized on a 2% agarose gel.
- Gel images were analyzed with ImageJ for relative intensity of each band
- 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).


Results

The expression of mtDNA polymerase and mtSSB but not mtDNA helicase genes are upregulated in the mitotically replicating tissues of Z∆MTS mutant larvae in comparison to wild type larvae of the same stage (figure 3a). Analysis of relative intensity of each band normalized to rp49 allowed to calculate that the expression of mtSSB and tamas is almost doubled in the Z∆MTS mutant larvae compared to the wild type (figure 3b)


Discussion

According to the results shown, the expression of mtDNA polymerase and single-stranded DNA binding protein is upregulated in RNZ∆MTS mutant larvae which provides an explanation to the fact that amount of mtDNA is increased. There is no observable difference in expression of mtDNA helicase and a possible explanation is that the mtHEL molecule might have a longer lifetime or higher processivity than other parts of mtDNA transcription machinery.

References

1. Spath, B, Canino, G., Marchfelder, A., 2007. tRNaseZ: the end is not in sight. Cell. Mol. Life Sci.
2. Vogel, A., Schilling, O., Spath, B., Marchfelder A., 2005. The tRNaseZ family of proteins: physiological functions, substrate specificity and structural properties. Biol. Chem.
3. Tavtigan, S.V., Simard, J., et al, (2001). A candidate prostate cancer susceptibility gene at chromosome 17p. Nat. Genetics
4. Haack TB, Kopajtich R, Freisinger P, Wieland T, Rorbach J, et al. (2013) ELAC2 Mutations cause a mitochondrial RNA processing defect associated with hypertrophic cardiomyopathy. Am J Hum Genet
5. Ojala,D., Montoya,J. and Attardi. G. (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature
6. Xie, X., Dubrovsky, E. (2015) Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression. NAR

Figures


Figure 1- RNaseZ and mitochondrial transcript processing. RNaseZ cuts at the 3’ end of each tRNA thus separating them from mRNAs and rRNAs


Figure 2- (a) qPCR analysis of mtDNA in WT and Z∆MTS wing discs, mtDNA values are normalized to nuclear DNA (b) citrate synthase activity, the marker for mitochondrial mass


Figure 3-Z∆MTS = mutant phenotype, WT = wild type. (a) RT-PCR results for the studied gene products. rp49 is a housekeeping gene. (b) Results of analysis of relative band intensity.


Figure 4-Model for the mechanism enabling the increase of mitochondrial mass and amount of DNA under condition of mitochondria failure. When mitochondrial function fails due to lack of RNaseZ, mitochondria sends signals to nucleus to upregulate tamas and mtSSB genes thus enabling production of more mitochondria


Summary

RNaseZ is a highly conserved gene, whose mutations were linked to prostate cancer and infantile hypertrophic cardiomyopathy in humans.To understand the role of this gene in such diseases the function of the protein encoded by this gene is studied using Drosophila flies as a model. When this protein is removed from mitochondria, the function of respiratory chain fails. In such cells the amount of mitochondria and mitochondrial DNA is drastically increased. The goal of this study is to identify the targets of retrograde signaling activating mitochondria biosynthesis.

Full Paper

Acknowledgments

I would like to give a sincere thanks to Dr. Rubin for his expert guidance through this project. I would also like to thank Catharina Grubaugh and Anthony Evans for their endless patience and all their time devoted to help us. I appreciate Dr. Dubrovsky’s lab for kindly providing the Drosophila stocks.


This document was last modified 05/16/2016.
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