Probing the Spatial Proteome

It is important to capture the inter-relationship of the localization of proteins and RNA in cells not only to understand basic cell biological mechanisms, but also how diseases arise from breakdown in this relationship. mRNA species are spatially located and translated in a highly controlled manner involving the interaction of cis- and trans- acting factors with RNA-binding proteins (RBPs). Aberrant translation of proteins in the wrong subcellular location gives rise to multiple diseases such as Spinal Muscular Dystrophy and Fragile X.

We have developed a suite of mass spectrometry methods, based around LOPIT (localization of organelle proteins using isotope tagging), to determine the three-dimensional organisation of the cellular proteome in eukaryote cells with high resolution. These methods are supported by robust machine learning approaches to interrogate complex patterns within these data. We have applied these methods to map protein re-localization upon perturbation. Intriguingly, we consistently observe that half the proteome cannot be discretely assigned to a single location, an observation supported by high-throughput immunocytochemistry.

To further explore the spatial localization of the transcriptome, we have created methods to map RNA and RBPs in cells. First, we have created OOPS, (orthogonal organic phase separation), a method that efficiently extracts protein. This highly flexible approach returns information on all RNA species greater than 200 bases, including lncRNAs. It can be used to identify protein-binding sites across the transcriptome and sites of RNA binding on proteins. We show that all long RNA in cells are bound by proteins and reveal previously undetected RBPs located across all membrane-bound organelles. We have applied OOPS to access changes in the RNA-binding proteome during the cell cycle, elucidating cellular mechanisms that are influenced by the dynamic interactions of RNA and protein.

Combining the above approaches, we are starting to build up a picture of the spatial inter-relationship of the proteome and transcriptome.