Project description:Spatiotemporal protein expression is mediated by active transport and local translation of mRNAs. Key factors of the transport machinery are RNA-binding proteins (RBPs) that recognise mRNAs as cargo for motor-based transport. However, a global view of transported mRNAs with cognate RBP binding sites is missing. Here, we cast a transcriptome-wide view on endosomal mRNP transport in Ustilago maydis by studying the newly identified endosomal RBP Grp1 and the key transport RBP Rrm4 in a comparative iCLIP approach. This uncovered thousand of cargo mRNAs bound by both RBPs.
Project description:Spatiotemporal protein expression is mediated by active transport and local translation of mRNAs. Key factors of the transport machinery are RNA-binding proteins (RBPs) that recognise mRNAs as cargo for motor-based transport. However, a global view of transported mRNAs with cognate RBP binding sites is missing. Here, we cast a transcriptome-wide view on endosomal mRNP transport in Ustilago maydis by studying the newly identified endosomal RBP Grp1 and the key transport RBP Rrm4 in a comparative iCLIP approach. This uncovered thousand of cargo mRNAs bound by both RBPs.
Project description:The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful new player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos to the TGN, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7—the logistical centerpiece of LE biology—as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.
Project description:A key function for RNA-binding proteins in orchestrating plant development and environmental responses is well established. However, the lack of a genome-wide view on their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) for genome-wide determining the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7. We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.
Project description:RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate several functions at once and likely impact the structural integrity of the large mRNP assemblies, these proteins often function in. Thus, the function of the RNA-binding activity of RBPs is often unknown. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric (MS) analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components. For functional analysis, we chose Npl3, for which we identified crosslinks in its two RNA recognition motifs (RRMs) as well as in the flexible linker connecting the two. Both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in each of these three regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutant strongly impairs recruitment of some mRNP components to chromatin and the incorporation of other mRNP components into nuclear mRNPs, establishing a function of Npl3 in mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of a nuclear mRNP component, an approach that can be applied to many RBPs in any RNA metabolic process.