Characterization of peroxisome-associated transcriptome

This is a pioneer genome-wide study of localization of mRNAs to peroxisomes. The findings suggest that translation of a subset of peroxiomal proteins and other cellular metabolic enzymes may be spatially regulated by directing their encoding mRNAs to the surface of peroxisomes. The study provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments. In this study we performed the genome-wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis. Two subcellular fractions were obtained from the mouse liver by centrifugation in iodixanol density gradient: 1) Peroxisomal (PX), 2) Mitochondrial/Lysosomal (ML). The membrane-enclosed peroxisomal fraction (PX-IP) was obtained by immuno-precispitation of the PX fraction with the PMP70-specific antibodies. RNA was extracted from each fraction. Additionally, the total cellular RNA was obtained from the whole cell extracts (T). Thus, four RNA sample types were obtained for gene expression analysis: T, ML, PX, PI. Three technical replicates of each sample ware analyzed with Illumina microarrays.