Short Hairpin RNA-mediated Silencing of PRC (PGC-1-related Coactivator) Results in a Severe Respiratory Chain Deficiency Associated with the Proliferation of Aberrant Mitochondria

PRC, a member of the PGC-1 coactivator family, is responsive to serum growth factors and up-regulated in proliferating cells. Here, we investigated its in vivo role by stably silencing PRC expression with two different short hairpin RNAs (shRNA1 and shRNA4) that were lentivirally introduced into U2OS cells. shRNA1 transductants exhibited nearly complete knockdown of PRC protein, whereas shRNA4 transductants expressed PRC protein at similar to 15% of the control level. Complete PRC silencing by shRNA1 resulted in a severe inhibition of respiratory growth; reduced expression of respiratory protein subunits from complexes I, II, III, and IV; markedly lower complex I and IV respiratory enzyme levels; and diminished mitochondrial ATP production. Surprisingly, shRNA1 transductants exhibited a striking proliferation of abnormal mitochondria that were devoid of organized cristae and displayed severe membrane abnormalities. Although shRNA4 transductants had normal respiratory subunit expression and a moderately diminished respiratory growth rate, both transductants showed markedly reduced growth on glucose accompanied by inhibition of G(1)/S cell cycle progression. Microarray analysis revealed striking overlaps in the genes affected by PRC silencing in the two transductants, and the functional identities of these overlapping genes were consistent with the observed mitochondrial and cell growth phenotypes. The consistency between phenotype and PRC expression levels in the two independent transductant lines argues that the defects result from PRC silencing and not from off target effects. These results support a role for PRC in the integration of pathways directing mitochondrial respiratory function and cell growth.