Mitochondrial and nuclear genomic response to loss of LRPPRC expression

Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remains challenging. Here, we introduce a genomic strategy to functionally characterize such genes, and apply it to LRPPRC (leucine-rich PPR-motif containing), a poorly studied gene that is mutated in Leigh Syndrome, French Canadian type (LSFC). We utilize RNAi to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. Using expression profiling, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA (mtDNA)-encoded mRNAs, but not the rRNAs, without affecting nuclear genes encoding mitochondrial proteins. We designed seven shRNAs targeting the LRPPRC cDNA sequence to silence its expression in MCH58 immortalized human fibroblasts. The LRPPRC expression level in these cells ranged from 9% to 100%. We demonstrated that knockdown cells carried stable silencing of the target gene and associated biochemical phenotypes. Our goal was to engineer stable knockdown cells which recapitulate the LSFC disease phenotype and subject them to expression profiling using Affymetric microarrays to identify genesets and biochemical pathways that are altered.