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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 multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA–protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that are implicated in cancer and regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains tocis-regulatory RNA elements. A structure of the RRM1-ZnF1 region in complex with RNA shows how the tandem domains cooperate to sandwich target RNA and specifically recognize a GG dinucleotide in a non-canonical fashion. While the RRM1-ZnF1 domains act as a single structural module, RRM2 is connected by a flexible linker and tumbles independently. However, all three domains participate in RNA binding and adopt a closed architecture upon RNA binding. Our data highlight how cooperativity and conformational modularity of multiple RNA binding domains enable the recognition of distinct RNA motifs, thereby contributing to the regulation of alternative splicing. Remarkably, we observe surprising differences in coupling of the RNA binding domains between the closely related homologs RBM5 and RBM10.HighlightsMultiple RNA binding domains enable differential recognition of distinct RNA motifs to regulate alternative splicingThe RRM1-ZnF1 domains of RBM5 mediate specific recognition ofcisregulatory RNA motifs to modulate alternative splicingRRM1-ZnF1 sandwich the target RNA for non-canonical recognition of a GG dinucleotideRRM1-ZnF1-RRM2 bind cooperatively to contiguouscis-regulatory motifs incaspase-2andNUMBpre-mRNAs

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 multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.

Description, ATP-driven conformational selection of Hsp90 conformations modulates the binding of clients and co-chaperones., Hsp90 is a molecular chaperone that interacts with a specific set of client proteins and assists their folding. The underlying molecular mechanisms, involving dynamic transitions between open and closed conformations, are still enigmatic. Combining nuclear magnetic resonance, small-angle x-ray scattering, and biochemical experiments, we have identified a key intermediate state of Hsp90 induced by adenosine triphosphate (ATP) binding, in which rotation of the Hsp90 N-terminal domain (NTD) yields a domain arrangement poised for closing. This ATP-stabilized NTD rotation is allosterically communicated across the full Hsp90 dimer, affecting distant client sites. By analyzing the interactions of four distinct clients, i.e., steroid hormone receptors (glucocorticoid receptor and mineralocorticoid receptor), p53, and Tau, we show that client-specific interactions with Hsp90 select and enhance the NTD-rotated state and promote closing of the full-length Hsp90 dimer. The p23 co-chaperone shifts the population of Hsp90 toward the closed state, thereby enhancing client interaction and processing.