Research Article: Comparative Sequence Analysis of the Non-Protein-Coding Mitochondrial DNA of Inbred Rat Strains

Date Published: December 7, 2009

Publisher: Public Library of Science

Author(s): Avinash Abhyankar, Hee-Bok Park, Giancarlo Tonolo, Holger Luthman, Laszlo Orban.

Abstract: The proper function of mammalian mitochondria necessitates a coordinated expression of both nuclear and mitochondrial genes, most likely due to the co-evolution of nuclear and mitochondrial genomes. The non-protein coding regions of mitochondrial DNA (mtDNA) including the D-loop, tRNA and rRNA genes form a major component of this regulated expression unit. Here we present comparative analyses of the non-protein-coding regions from 27 Rattus norvegicus mtDNA sequences. There were two variable positions in 12S rRNA, 20 in 16S rRNA, eight within the tRNA genes and 13 in the D-loop. Only one of the three neutrality tests used demonstrated statistically significant evidence for selection in 16S rRNA and tRNA-Cys. Based on our analyses of conserved sequences, we propose that some of the variable nucleotide positions identified in 16S rRNA and tRNA-Cys, and the D-loop might be important for mitochondrial function and its regulation.

Partial Text: Mitochondria are the major energy producers in eukaryotic cells. Over millions of years of coexistence and coevolution, mitochondria have lost a considerable amount of their genome to the eukaryotic nuclear DNA [1], [2]. The mammalian mitochondrial DNA (mtDNA) encodes 37 genes, 13 of which form essential subunits of four mitochondrial respiratory chain complexes. The remaining genes for these complexes are encoded by the nuclear genome. Consequently, mitochondrial biogenesis, and hence function, needs an elaborate coordination of nuclear and mitochondrial gene expression [3], [4]. Apart from several ultra-short intergenic non-coding regions, mtDNA possesses a large non-coding D-loop that harbors regulatory regions for transcription and replication. The D-loop regulates mitochondrial replication and transcription in accordance with the energy demands, while the mitochondrial rRNAs and tRNAs ensure fulfillment of this task. Having its own genetic code different from the nuclear genetic code, mitochondria need their own protein biosynthesis system in the form of the mitochondrial ribosome (mitoribosome) built around 12S rRNA and 16S rRNA. The mitoribosome is responsible for the biosynthesis of the 13 proteins coded by the mtDNA and translates them with the help of 22 tRNAs also encoded by mtDNA. The non-protein-coding regions of the mtDNA are indispensable for cellular energy homeostasis, and genetic variation in these regions could have metabolic and fitness consequences. Since the protein-coding and the non-protein-coding regions of mtDNA serve different purposes – function and regulation of function – the variation pattern and the evolutionary pressures are expected to be different. Furthermore, the relative significance of coding sequence variation compared to the regulatory sequence variation, from an evolutionary perspective, remains poorly understood [5]. For this reason we investigated the protein-coding and the non-protein-coding regions separately. Here we present a molecular evolutionary analysis of the RNA genes and the D-loop of the rat mitochondrial genome. Information from 27 complete Rattus norvegicus mtDNA sequences was used.

Mitochondrial DNA encodes few but essential components of the respiratory chain complexes I, III, IV and V. The two ribosomal RNAs provide a scaffold for the mitochondrial ribosomal proteins (MRPs). The mammalian mitoribosome has significantly reduced RNA content as compared to its bacterial counterpart; this reduction is accounted for by an increase in the number of MRPs [13], [14]. This reduction exerts strict structural constraints on the ribosomal RNAs for efficient and accurate function. In bacteria and archea the ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor by binding mRNA [15], [16]. The L1 binding domain in the mammalian mitochondrial 16S rRNA was found to be highly conserved [17]. According to our analysis, only one of the observed variable positions in the rat 16S rRNA (position 2170) is highly conserved and might be of functional importance due to its close proximity to the L1 binding domain (Figure 1).



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