Browsing Upstate Medical University by Subject "based approach"
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A trancriptomics based approach reveals the functional consequences of RNase MRP RNA mutations in yeast.RNase MRP is a eukaryotic ribonucleoprotein complex involved in multiple cellular functions that includes ribosomal RNA processing, primer generation for mitochondrial DNA replication and degradation of cell cycle related mRNAs. In Saccharomyces cerevisiae, the RNA component of RNase MRP is encoded by NME1. We have performed random deletion mutagenesis of RNase MRP RNA gene and isolated a mutation, nme1-91, that causes temperature sensitive growth defect on glycerol media. RNA analysis of nme1-91 showed that this mutant is mildly deficient in the 5.8S rRNA processing function of RNase MRP. Growth analysis and northern blotting of RNase MRP RNA mutations generated based on nme1-91 allele suggested that 3’-end nucleotide sequences of the nme1-91 allele contribute to its phenotype. Highcopy suppression screen identified tRNA modification gene NCS6 as a suppressor of nme1-91. Additionally, primary mode of suppression by NCS6 was found to be non-mitochondrial since NCS6 partially suppressed the nme1-91 phenotype on fermentable carbon source. Strains carrying a deletion of NCS6 in combination with nme1-91 showed a synthetic sick phenotype. Polysome profile analysis of nme1-91 revealed that 80S monosomal fraction accumulates in this mutant. Differential gene expression analysis of nme1-91 by RNAseq indicated that rRNA processing and cell cycle related genes become mis-regulated due to this mutation. A similar high-throughput sequencing based approach was also employed to investigate the transcriptional basis of positive genetic interactions between components of RNase MRP and nonsense-mediated decay pathway. A yeast strain bearing the nme1-P6 mutation in the RNA component of RNase MRP exhibits temperature-sensitive growth defect. This phenotype can be suppressed by deletion of NMD components. Differential gene expression analysis identified several mis-regulated biological processes in nme1-P6 and Δupf1 strains. Comparative transcriptomic analysis suggested that suppression of nme1-P6 phenotype by Δupf1 is accompanied by large shift in gene expression pattern towards Δupf1 strain. Moreover, the majority of direct targets of NMD were not down-regulated in nme1-P6 indicating that the effect of NMD on nme1-P6 might be due to increased degradation on mRNAs that are not targeted by NMD in normal conditions. Taken together, these results show that mutations of RNase MRP RNA can modulate diverse biological processes.