RNA Isolation from Single Living Cells using AFM

Abstract

Many protein-RNA complexes exist within cells and have various functions, including shuttling mRNA from the site of transcription to specific cellular regions. Often, these protein-RNA complexes consist of multiple mRNAs of which some are known and others remain unknown. The traditional method used to isolate protein-mRNA complexes is immunoprecipitation (IP). IP involves lysing a population of cells and isolating the protein-mRNA complexes using antibodies specific to the protein to pulldown the corresponding protein-mRNA complex of interest. We demonstrate the use of AFM as a complementary analysis tool for studying protein-RNA complexes in single living cells. While IP homogenizes the composition of protein-mRNA complexes from the cellular population under investigation and represents an average, the AFM can interrogate not only a single cell but also subcellular regions of a single live cell, allowing stratification of protein-mRNA complexes from different regions of the cell. The result is the ability to spatially discriminate different populations of mRNAs that may be carried by the shuttling protein. To demonstrate extraction of a protein-mRNA complex, we selected the shuttling protein, Zipcode Binding Protein 1 (ZBP1) and its known associated mRNA, beta-actin, as a model system. Rat fibroblasts expressing a fusion protein, ZBP1-mCherry, were visualized using the fluorescent microscope of the AFM and then the cells of interest were punctured with an AFM tip conjugated with antibodies to ZBP1. Subsequently, the AFM tip was collected and analyzed for beta-actin mRNA using RT-PCR. BioAnalyzer results confirmed the extraction of beta-actin mRNA. This work shows that the AFM can be used as a tool to extract protein-RNA complexes from different regions of single living cells, potentially expanding the cell biologist's toolset.