Gimap3 Regulates Tissue-Specific Mitochondrial DNA Segregation

Peer reviewe

MtDNA copy number regulation in the spleen has no effect on mtDNA segregation.

<p>MtDNA copy number relative to nuclear DNA was measured in the spleen of three month F2 (BALB/c X CAST/Ei) mice with or without mtDNA selection. Data are presented as means ± SD (+ mtDNA selection, n = 32; - mtDNA selection, n = 13).</p

A chromosome 6 locus significantly affects mtDNA segregation in hematopoietic tissues.

<p>Genome-wide linkage analysis from 168 F2 (BALB/c X CAST/Ei) mice searching for loci regulating the loss of mtDNA selection. Only the chromosome 6 locus was significantly linked to this phenotype (LOD 4.6, genome-wide p = 0.007 with 10,000 permutations).</p

<i>Gimap3</i> gene structure and protein sequence in BALB/c and CAST/Ei mouse strains.

<p>A. Exon structure and splicing of <i>Gimap3</i>. An AUG start codon is present in both exon 3 and exon 4. In exon 4, upstream of AUG start codon is a stop codon. In the BALB/c allele all 5 exons are spliced together, so translation of the mature protein initiates at the second AUG start codon, with a predicted size of 34 kDa. In the CAST/Ei allele, a G-A transition in the splice acceptor site of exon 4 prevents its splicing into the mature mRNA, so exon 4 is missing and translation initiates from the first AUG start codon, predicting a protein of 41 kDa. B. Alignment of the <i>Gimap3</i> exon 4 and flanking intronic sequence from 5 <i>Mus musculus domesticus</i> strains, all of which are indistinguishable in the phenotype for mtDNA segregation compared to the <i>Mus musculus castaneus</i> CAST/Ei strain. C. ClustalW alignment of CAST and BALB Gimap3 protein sequences.</p

Ectopic expression of <i>Gimap3</i> has no effect on mtDNA segregation.

<p>A. Relative fitness of NZB mtDNA in the liver and kidney of F2 transgenic littermates positive or negative for the CAST/Ei <i>Gimap3</i> cDNA. Data are presented as means ± SD (transgene negative, n = 16; and positive, n = 17). B. Cultured heteroplasmic murine embryonic fibroblasts were transduced with BALB <i>Gimap3</i> or CAST <i>Gimap3</i> containing an N-terminal HA tag in pBABE, or with empty vector (pBABE). Cells were grown continuously in culture for 1 month. The change in NZB heteroplasmy in the bulk culture was determined comparing the level after 1 month of culture to the initial level before retroviral transduction.</p

Transgenic expression of CAST/Ei <i>Gimap3</i> cDNA in heteroplasmic mice slows the rate of splenic mtDNA segregation.

<p>A. CAST/Ei <i>Gimap3</i> transgene expression across a number of mouse tissues (B-brain; L-lung; H-heart; S-spleen) driven off the ROSA26 promoter. Endogenous <i>Gimap3</i> expression from BALB/c or CAST/Ei spleen was loaded as a control. Equal amounts of total RNA were amplified by RT-PCR under the same conditions in each tissue. Beta-actin was used as a control. B. CAST/Ei <i>Gimap3</i> transgene expression in three-month-old mice significantly slows down the mean rate of mtDNA segregation in the spleen compared to littermate controls and the BALB/c heteroplasmic mouse model (ANOVA, p = 0.0011). Data are presented as a scatter plot with means indicated (bar). Transgene negative (n = 16) and positive (n = 17); BALB/c (n = 23).</p

MtDNA segregation in hematopoietic tissues of 12-month-old heteroplasmic F2 (BALB/c X CAST/Ei) mice.

<p>A representative profile of mtDNA heteroplasmy levels in hematopoietic (spleen, peripheral blood, and bone marrow) and neutral tissues (heart, brain and skeletal muscle) from four 12-month-old F2 (BALB/c X CAST/Ei) mice illustrates the mtDNA segregation phenotypes. Mice were classified as having no (-) mtDNA selection if the % NZB mtDNA in hematopoietic tissues was similar to neutral tissues or having (+) mtDNA selection. Data is presented from mice with high (>60%) or moderate (35%) levels of NZB heteroplasmy in their neutral tissues.</p

Chromosome 6 genes located between 37–49 Mb with a role in mitochondrial biology (GO:0005739).

<p>Chromosome 6 genes located between 37–49 Mb with a role in mitochondrial biology (GO:0005739).</p

Enrichment of the CAST/Ei <i>Gimap3</i> allele in three-month-old F2 mice with no mtDNA selection.

<p>Three-month-old F2 (BALB/c X CAST/Ei) mice (n = 145) were classified into two groups, presence (+) (n = 88, 0.61) or absence (-) (n = 57, 0.39) of mtDNA selection, and then genotyped for their <i>Gimap3</i> alleles. Distributions were compared to the expected Mendelian ratios by Chi-square analysis (p = 0.00033).</p

Gimap3 and Gimap5 protein sequences.

<p>A. ClustalW alignment of BALB Gimap3 and Gimap5 protein sequences. B. ClustalW alignment of BALB and CAST Gimap5.</p