Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3 and 5 UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.neuron ͉ transcriptome-induced phenotype remodeling ͉ single cell ͉ Waddington I n multicellular organisms, all cells contain nearly identical copies of the genome but exhibit drastically different phenotypes. Even a single neuron has a set of phenotypic characteristics that distinguish it from other neurons as well as other cell types, such as the nearby astrocytes. Indeed, as Waddington proposed in his classical epigenetic landscape model, genetically predetermined cells can follow any specific permitted trajectories that eventually lead to different cellular phenotypes (1). From this point of view, the genome serves as a repository of dynamic control information whose state can be reprogrammed to match the stable phenotypic states.Emerging evidence has demonstrated the reversibility and flexibility of the cellular phenotype. Gurdon et al. first showed that the ability to obtain fertile adult male and female frogs by injecting endoderm nuclei into enucleated eggs (2). This result not only forms the foundation of the field in nuclear transplantation but also provides evidence that the cytoplasmic components of a differentiated cell can support nuclear reprograming. Generation of induced pluripotent stem (iPS) cells by transfection of transcription factors into dividing fibroblasts (3), followed by cell selection, represents a new strategy to globally revert a mature cell into a different cell type (4-9). The need for redifferentiation of these embryonic stem cell-like-iPS cells into desired cell types adds a layer of complexity that is difficult to control (10, 11). Nevertheless, studies of nuclear reprogramming from genomic and epigenetic modification, as seen from so...

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