Alternative splicing of ndufs2 and cox10 oxphos genes during aging




Yuhan Hao

Introduction

Age-related macular degeneration (AMD) is a major cause of blindness which lead to the deterioration of the center of the retina, among the elderly period. Nearly 40% of people over 75 years old, have some pathologic signs of AMD [1]. Many lines of evidence suggest that mitochondrial disorders in eyes contribute to AMD, and mitochondrial disorders are common in other neurodegeneration diseases, such as Parkinson’s disease and Alzheimer disease [2,3]. Mitochondrial disorders are clinical phenotypes associated with mitochondrial dysfunction, and abnormalities of OXPHOS.

OXPHOS is responsible for 90% of ATP production in a respiring cell. It has five multi-subunit complexes, the respiratory chain complexes, and two additional electron carriers. It is controlled on the genetic level by two distinct genomes: the mitochondrial genome and the nuclear genome. The mitochondrial genome of nearly 16.6 kb, encodes 13 subunits of complex I, III, IV and V [4].

Deficiencies of OXPHOS are able to lead direct and indirect changes in metabolic homeostasis, which includes concentrations of ROS, Ca2+, and ratios of ADP/ATP, and NAD/NADH [5]. Transcriptional response of OXPHOS genes, both nDNA and mtDNA, is often used to adapt these metabolic changes. Besides, OXPHOS genes have a feature of co-expression. And it is evident from studies of transcriptome analysis across different species. Particularly, in mice, co-expression of OXPHOS genes are studied among various tissues [6]. What is more, it is found that OXPHOS genes are likely to have co-regulations. There may be the likely core promoters for most OXPHOS genes and assembly factors [7].

Complex I deficiency is the most common cause of the mitochondrial disease. NDUFS2 is a constitutive component of the core of complex I, considered to be essential for electron transfers. It is encoded by nDNA and has thirteen exons. The NDUFS2 mutation may lead to dysfunction of OXPHOS complex I where the mutation caused a catalytic defect .This mutation may have relation with Leigh syndrome [8]. Leigh syndrome is a severe neurological disorder disease with features of progressive degeneration of mental and movement abilities. 

Cytochrome c oxidase(COX), embedded in the inner mitochondrial membrane, is in the terminal enzyme of electron transport chain. It catalyzes transfer of electrons from cytochrome c to oxygen [9]. Deficiency of COX is one of the most common metabolic reasons associated with respiratory chain defects and it is associated with various mitochondrial and neurodegenerative diseases [10,11]. COX10, one of subunits of COX, is heme-O-farnesyl transferase and also an assembly factor for COX in complex IV. It is encoded by nDNA and has seven exons. It is reported that mice lacking COX10 in skeletal muscle exhibited a progressive mitochondrial disease phenotype [12].

Materials and Methods

Animal
Mice were provided by Finnemann’s lab, Department of Biological Sciences, Fordham University.Young, middle-aged mice were three and six months old, respectively. Mice were sacrificed by CO2 and eyeballs were isolated immediately and immersed in Davidson's fixative. Retina and eyecups were separated and then were grind.
RNA extraction
RNA was extracted from young mice retina, eyecups and middle-aged mice retina and eyecups using RNeasy® Plus Mini Kit (QIAGEN), according to the manufacturer’s instructions.
Primers
Seven pairs of primers were designed for this experiment. 4 pairs of primers were used to cover gene ndufs2 and the other primers were used to cover gene cox10. Most expected size of product is about 500 bp. The Tm of most of the primers is near 60°C.
Reverse Transcriptase-PCR (RT-PCR)
RT-PCR was performed using QIAGEN® One-Step RT-PCR Kit following instructions.Ten nanograms of RNA was amplified in 20 ul RT-PCRs. Temperature cycles as follow: one cycle of 50°C for 30min and 95°C for 15min, 94°C for 30 s, 57°C for 30s, and 72°C for 30s, and a final extension of 72°C for 10 min followed by a final hold at 4°C. Cycle number was 50.
Electrophoresis
5 ul of loading dye was added to each RT-PCR product. 5 ul of each product was then added to a 1% agarose gel, and electrophoresis was performed at 160 V. Band intensities were visualized by ethidium bromide in a UV trans-illuminator (BioRad). 100 bp marker was used to to measure the size of bands.
Gel extraction and sequencing
The target products was extracted by QIAquick Gel Extraction Kit(QIAGEN) following the manufacturer's instructions and subsequently sequenced by GENEWIZ® in order to identify PCR products.

Results

I. No alternative variants were found in NDUFS2
In order to detect alternative splicing of NDUFS2, four pairs of primers were used for ndfus2, from exon1 to exon3 and from exon3 to exon7, from exon6 to exon10, and from exon9 to exon13. They overlapped all exons of ndufs2. If alternative splicing existed, two or more bands were observed from electrophoresis gel. But no alternative splicing was observed in ndufs2 (Fig. 1). Only expected bands were obtained. And expression level of ndufs2 were also constant during aging (Fig. S1). The result indicated that alternative splicing of NDUFS2 might not be affected by aging.

II. Spliced variants of COX10 were observed
In order to detect alternative splicing of COX10, three pairs of primers were used for cox10, from exon1 to exon3 and from exon2 to exon4, and from exon3 to exon7. No alternative splicing were detected with first two pairs of primers(Fig. S2). However, it was surprising that three bands in young mice and two bands in middle-age mice were detected within primers from exon3 to exon7 (Fig. 2).Variant C was only found to be expressed strongly in young mice. Three bands were purified and sent out for sequencing and were confirmed that they were three variants of COX10 by blast (Fig. 3). The sequences of three bands were aligned and analyzed by blast and clustalW. It was found that variant A was expected results, in which no exons were spliced out and variant B resulted from part of exon6, 45 bp was spliced out. No reading frame shift happened. Conserved region for alternative splicing acceptor site were also analyzed and compared with variant A (Fig. 4). Polypyrimidine tract, T and C rich region, and 3’ end AG were necessary for acceptor site, and they both found spliced end region on variant B. In variant C, exon6 was entirely spliced out, without read frame shift. Variant B and variant C were not reported in mouse database, but variant C matched a homologous variant in human database. Variant B has not been reported in any database.

Discussion

This project found expressions of three variants of COX10, in mice retina and eyecups, changed with aging. Transmembrane domains were predicted by TMHMM Server. Variant A has nine putative transmembrane domains.The spliced region in variant B is located in two transmembrane domains; and variant C lacks four transmembrane domains. It is surprising that variant C is expressed strongly in young mice eyes. The biological meaning of these spliced variants is not understood clearly. Variant B and C are not found in mouse genomic plus transcript database in NCBI. Variant C matched a homologous variant in the human database, variant B, however, has not been reported in any database. Western blot of variant B and variant C are still required, because those two variants may not be translated into proteins.

References

[1] Hughes, Anne E., et al. "A common CFH haplotype, with deletion of CFHR1 and CFHR3, is associated with lower risk of age-related macular degeneration."Nature genetics 38.10 (2006): 1173-1177.
[2] Wallace DC “Mitochondrial DNA mutations in diseases of energy metabolism.” J Bioenerg Biomembr 26 (1994) : 241–250.
[3] Kenney,. et al. "Mitochondrial DNA variants mediate energy production and expression levels for CFH, C3 and EFEMP1 genes: implications for age-related macular degeneration." PLoS One 8.1 (2013): e54339.
[4] G. Cannino, C.M. Di Liegro, A.M. Rinaldi, “Nuclear–mitochondrial interaction”, Mitochondrion 7 (2007): 359–366.
[5] Reinecke, Fimmie, Jan AM Smeitink, and Francois H. Van Der Westhuizen. "OXPHOS gene expression and control in mitochondrial disorders." Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1792.12 (2009): 1113-1121.
[6] Mootha VK, Bunkenborg J, et al “Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria.” Cell, 115 (2003):629-640.
[7] van Waveren, Corina, and Carlos T. Moraes. "Transcriptional co-expression and co-regulation of genes coding for components of the oxidative phosphorylation system." BMC genomics 9.1 (2008): 18.
[8] Ngu, Lock Hock, et al. "A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome."Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1822.2 (2012): 168-175.
[9] Capaldi, R.A. “Structure and assembly of cytochrome c oxidase.” Arch. Biochem. Biophys., 280 (1990): 252–262.
[10] Darin, N., Moslemi, A.R., Lebon, S., et al “Genotypes and clinical phenotypes in children with cytochrome-c oxidase deficiency.” Neuropediatrics, 34 (2003): 311–317.
[11] Comi, G.P., Strazzer, S., Galbiati, S. and Bresolin, N. “Cytochrome c oxidase deficiency.” Int. Rev. Neurobiol., 53 (2002): 205–240.
[12] Diaz, Francisca, et al. "Mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency." Human molecular genetics 14.18 (2005): 2737-2748.

Figures


Figure 1- RT-PCR results for ndufs2 using four pairs of primers. C is control group.YE is young mouse eyecup; and YR is young mouse retina. ME is middle age mouse eyecup; and MR is middle age mouse retina.All expected size of bands is about 500 bp. No alternative splicing was found.


Figure 2- RT-PCR results of cox10 from exon3 to exon7. Size of band A and band B was near 500 bp. Size of band C was near 200 bp. MR also had band C, but it was very weak.


Figure 3- Alternative pathways of COX10. Here were three variants of COX10. Variant A was normal variant. Part of exon6, 45 bp, was in Variant B and reading frame was not moved. The whole exon6 was spliced out in variant C.


Figure 4- Comparison of alternative splicing acceptor site of variant A and variant B. Arrows indicate end of acceoptor sites. Lower case letters were spliced out sequences; and upper case letters were sequences contained in variants. Both variants had polypyrimide tract and 3’ end AG in acceptor sites.


Abstract
AMD is associated with mitochondrial disorders, and abnormalities in the oxidative phosphorylation system (OXPHOS) contributed to mitochondrial dysfunction. Alternative RNA splicing of two genes, ndufs2 from complex I and cox10 from complex IV, were studied in retina and eyecups of young and middle-aged mice. Two novo splice variants of COX10,1098 bp and 1287 bp were detected in retina and eyecup in the young mice. RT-PCR products were found to have 45 bp and 234 bp spliced out. The 45 bp deleted variant, has not been reported in any database.

Full Paper

Acknowledgments

I would like to thank Dr. Finnemann for providing young mice and middle-aged mice and teaching me how to dissect eyes. I would like to thank Kate Reid and Catherina Grubaugh for their fantastic work as Teaching Assistants and for their never-ending patience and efforts they put in to make this project undergo successfully. Finally, I would like to thank Dr. Rubin for his guidance and for making this project possible.


This document was last modified 05/12/2015.
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