Dissecting the role of shuttling mRNA binding proteins in nucleocytoplasmic RNA transport

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All mRNA molecules are subject to posttranscriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. The recent introduction of deep sequencing technologies has enabled the development of new methods for broadly mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites in human cells. Therefore, it is now possible to resolve interdependencies and redundancies of binding of RBPs and ribonucleoprotein particles (RNPs) to mRNA molecules and evaluate the contribution of these interactions to gene regulation in the context of organismal development or normal and disease states. Uncovering the (redundant) sequence elements and the regulatory importance of RNA-RBP interactions will be critical to interpret human genetic variation in regulatory RNA regions and non-coding transcripts increasingly uncovered by genome-wide deep sequencing. The inspiration to study mRBPs in nucleocytoplasmic transport came from my unexpected observation that exportin 5 (XPO5), a karyopherin-type transporter, previously thought to be exclusively responsible for the nucleocytoplasmic transport of pre-microRNAs, bound more than 2,000 mRNAs at approx. 8,000 defined binding sites residing in regions predicted to form stable stem-loops. These results indicate that delineating the RNA binding properties of the nucleocytoplasmic transporters will yield important insights in the redundancies and specificities of RNA transport. The major goals of this application are to identify and characterize the interaction network of mRNA-binding transport proteins and their RNA targets at a sequence and functional level and to establish experimental models that relate these features to RNA transport processes and PTGR. I will systematically study the members of the human transportin/karyopherin/exportin/Ran-binding proteins (KAP), and the nuclear export factor TAP (NXF) proteins, and at the same time identify transport adapters or factors important during cargo loading and release. I will address this challenging problem through three specific aims: Aim 1: Comprehensive identification of target RNA sites for nucleocytoplasmic transport proteins. RNA targets transported and regulated by human KAP and NXF proteins will be identified and the target sites and binding elements will be determined by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP). Integrated annotation of binding sites on transcripts across libraries will allow for the identification of targets for combinatorial and redundant regulation. Aim 2: Identification of adapter RNA binding proteins interacting with nucleocytoplasmic transporters and involved with the transport of subsets of RNA. Unknown RBPs interacting with transporters will be identified either by targeted mass-spectrometry from silver-stained gels, or globally by SILAC based proteomics. The RNA targets and binding sites for newly identified proteins will be globally determined by PAR-CLIP. Aim 3: Development of assay systems to interrogate the transporter interaction network and elucidation of its role in normal and disease states. Reporter RNAs will be generated to assess the specific role of RBPs in nucleocytoplasmic transport. Visualize abundant transported mRNAs by RNA-FISH in diseased and healthy FFPE tissue. Bioinformatically mine existing disease-relevant expression and genome variation data under specific consideration of the redundancies in RBPs and multiple site occurrences in mRNAs uncovered by this study to identify mRNA targets that can be used to develop reporter RNAs.