BioProject

PRC, a member of the PGC-1 coactivator family, is responsive to serum growth factors and up regulated in proliferating cells. More...

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 (shRNA#1 and shRNA#4) that were lentivirally introduced into U2OS cells. ShRNA#1 transductants exhibited nearly complete knockdown of PRC protein whereas shRNA#4 transductants expressed PRC protein at approximately 15 percent of the control level. Complete PRC silencing by shRNA#1 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, shRNA#1 transductants exhibited a striking proliferation of abnormal mitochondria that were devoid of organized cristae and displayed severe membrane abnormalities. Although shRNA#4 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 G1/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. Overall design: U2OS cells were infected with lentivirus expressing either a control short hairpin RNA, or two independent short hairpin RNAs (shRNA1 and shRNA4) targeting PRC mRNA, and stable clones were isolated under blasticidin selection. Total RNA was isolated from U2OS control shRNA, shRNA1, and shRNA4 transductants using TRIzol (Invitrogen) and DNase treated with the TURBO DNA-free kit (Ambion). RNA samples were further purified using the RNeasy MinElute Cleanup kit (Qiagen). Integrity of the RNA was evaluated using the Agilent 2100 BioAnalyzer. 500 ng of RNA was used to perform in vitro transcription in the presence of biotin UTP with the Illumina TotalPrep RNA amplification kit (Ambion). The amplified, labeled RNA (1.5 μg) was then hybridized in triplicate to an Illumina Whole-Genome Sentrix Human-6 v2 Expression BeadChip, and detected according to the Illumina user manual. Less...