Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands.

U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon-intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

A transient α-helix in the N-terminal RNA recognition motif of polypyrimidine tract binding protein senses RNA secondary structure.

The polypyrimidine tract binding protein (PTB) is a multi-domain protein involved in alternative splicing, mRNA localization, stabilization, polyadenylation and translation initiation from internal ribosome entry sites (IRES). In this latter process, PTB promotes viral translation by interacting extensively with complex structured regions in the 5'-untranslated regions of viral RNAs at pyrimidine-rich targets located in single strand and hairpin regions. To better understand how PTB recognizes structured elements in RNA targets, we solved the solution structure of the N-terminal RNA recognition motif (RRM) in complex with an RNA hairpin embedding the loop sequence UCUUU, which is frequently found in IRESs of the picornovirus family. Surprisingly, a new three-turn α3 helix C-terminal to the RRM, folds upon binding the RNA hairpin. Although α3 does not mediate any contacts to the RNA, it acts as a sensor of RNA secondary structure, suggesting a role for RRM1 in detecting pyrimidine tracts in the context of structured RNA. Moreover, the degree of helix formation depends on the RNA loop sequence. Finally, we show that the α3 helix region, which is highly conserved in vertebrates, is crucial for PTB function in enhancing Encephalomyocarditis virus IRES activity.

Combining Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) Spectroscopy for Integrative Structural Biology of Protein-RNA Complexes.

Deciphering complex RNA-protein interactions on a (near-)atomic level is a hurdle that hinders advancing our understanding of fundamental processes in RNA metabolism and RNA-based gene regulation. To overcome challenges associated with individual structure determination methods, structural information derived from complementary biophysical methods can be combined in integrative structural biology approaches. Here, we review recent advances in such hybrid structural approaches with a focus on combining mass spectrometric analysis of cross-linked protein-RNA complexes and nuclear magnetic resonance (NMR) spectroscopy.

Spin labelling for integrative structure modelling: a case study of the polypyrimidine-tract binding protein 1 domains in complexes with short RNAs.

A combined method, employing NMR and EPR spectroscopies, has demonstrated its strength in solving structures of protein/RNA and other types of biomolecular complexes. This method works particularly well when the large biomolecular complex consists of a limited number of rigid building blocks, such as RNA-binding protein domains (RBDs). A variety of spin labels is available for such studies, allowing for conventional as well as spectroscopically orthogonal double electron-electron resonance (DEER) measurements in EPR. In this work, we compare different types of nitroxide-based and Gd(iii)-based spin labels attached to isolated RBDs of the polypyrimidine-tract binding protein 1 (PTBP1) and to short RNA fragments. In particular, we demonstrate experiments on spectroscopically orthogonal labelled RBD/RNA complexes. For all experiments we analyse spin labelling, DEER method performance, resulting distance distributions, and their consistency with the predictions from the spin label rotamers analysis. This work provides a set of intra-domain calibration DEER data, which can serve as a basis to start structure determination of the full length PTBP1 complex with an RNA derived from encephalomycarditis virus (EMCV) internal ribosomal entry site (IRES). For a series of tested labelling sites, we discuss their particular advantages and drawbacks in such a structure determination approach.

Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins.

Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron-electron resonance (DEER) EPR measurements were performed for both the nitroxide-nitroxide and Gd(III)-nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²âº release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.