Genome-Scale Identification of Membrane-Associated Human mRNAs

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Abstract
The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either “membrane-associated/secreted” (MS) or “cytosolic/nuclear” (CN). Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases. An important goal of current biological research is annotation of human genes with relevant descriptors and properties. One critical property of interest to biologists and medical researchers is the subcellular localization of gene products, as this affects a protein's biological role and our ability to use it as a therapeutic target. This study used a microarray-based functional genomic method that allows rapid, large-scale identification of subcellular localization, enabling the authors to annotate the localization of thousands of previously uncharacterized human gene products. The authors then provide an example of how these data can be used by applying them to the search for tumor-specific markers. Using data from hundreds of DNA microarray profiles of global gene expression patterns in tumors and normal tissues, they identify candidate genes encoding membrane-associated and secreted proteins that are highly overexpressed in tumors and that might therefore be particularly good targets for diagnostic tests or molecular therapies. Diagnostic tests based on these markers could potentially enable cancers to be detected earlier than is currently possible, and molecular therapies targeting the products of these genes could have high specificity for the corresponding cancers.