In the majority of cells, 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) (Harvey, WIRE RNA, 2018). Aberrant translation of proteins in the wrong subcellular location can give rise to multiple disease states such as Spinal Muscular Dystrophy, Fragile X, and autism.
We have developed a suite of methods, based around LOPIT (localization of organelle proteins using isotope tagging), to determine the three dimensional organisation of the proteome in eukaryote cells with high resolution (Mulvey, Nature Protocols (2017)). We have applied these methods to many different biological systems, including mapping protein relocalization upon perturbation. Intriguingly, we consistently observe that up to half the proteome cannot be discretely assigned to a single localisation. This observation has been recently supported by comparing two very different approaches to map proteins to their subcellular niches (Thul, Science (2017)).
To gain insight into the relationship between the steady state location of the translatome and its control though RBP interaction, we have built upon the above methods to create a highly efficient capture method, OOPS (orthogonal organic phase separation) which enriches UV crosslinked RNA and protein. This method samples interface between aqueous and organic solutions to enrich RBPs in a manner which is compatible with downstream proteomics and RNA sequencing. OOPS is able to recover all the protein bound (PBR) and free RNA in an unbiased way, and from the same sample, all the crosslinked RBP and free proteins. We have applied this method to multiple biological systems identifying the majority of previously identified RBPs but also, novel groups of RBPs. Sequencing of the PBR indicates that essentially all long RNAs in the cell are bound by proteins, enabling the identification of protein binding sites of the full transcriptome via read coverage.
We have also adapted the LOPIT method to be compatible with capturing the spatial location of RBP and PBRs to create spatial maps.
These methods will be reviewed and the implications of spatial translation on cellular mechanisms reviewed.