Cross-linking Data Research Articles

Simplified Protocol for Cross-linking Mass Spectrometry Using the MS-Cleavable Cross-linker DSBU with Efficient Cross-link Identification.

Chemical cross-linking combined with mass spectrometry (MS) is a powerful approach to identify and map protein-protein interactions. Its applications support computational modeling of three-dimensional structures and complement classical structural methodologies such as X-ray crystallography, NMR spectroscopy, and electron microscopy (EM). A plethora of cross-linkers, MS methods, and data analysis programs have been developed, but due to their methodological complexity application is currently reserved for specialized mass spectrometry laboratories. Here, we present a simplified single-step purification protocol that results in improved identifications of cross-linked peptides. We describe an easy-to-follow pipeline that combines the MS-cleavable cross-linker DSBU (disuccinimidyl dibutyric urea), a Q-Exactive mass spectrometer, and the dedicated software MeroX for data analysis to make cross-linking MS accessible to structural biology and biochemistry laboratories. In experiments focusing on kinetochore subcomplexes containing 4-10 subunits (so-called KMN network), one-step peptide purification, and enrichment by size-exclusion chromatography yielded identification of 135-228 non-redundant cross-links (577-820 cross-linked peptides) from each experiment. Notably, half of the non-redundant cross-links identified were not lysine-lysine cross-links and involved side chains with hydroxy groups. The new pipeline has a comparable potential toward the identification of protein-protein interactions as previously used pipelines based on isotope-labeled cross-linkers. A newly identified cross-link enabled us to improve our 3D-model of the KMN, emphasizing the power of cross-linking data for evaluation of low-resolution EM maps. In sum, our optimized experimental scheme represents a viable shortcut toward obtaining reliable cross-link data sets.

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator.

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization.

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data.

This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.

CircSMARCA5 Inhibits Migration of Glioblastoma Multiforme Cells by Regulating a Molecular Axis Involving Splicing Factors SRSF1/SRSF3/PTB.

Circular RNAs (circRNAs) have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM) pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5): it was significantly downregulated in GBM biopsies as compared to normal brain tissues (p-value < 0.00001, student’s t-test), contrary to its linear isoform counterpart that did not show any differential expression (p-value = 0.694, student’s t-test). Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma’s histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs), specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1), a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE). Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3) RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD) substrate. Interestingly, SRSF3 is known to interplay with two other splicing factors, polypyrimidine tract binding protein 1 (PTBP1) and polypyrimidine tract binding protein 2 (PTBP2), that positively regulate glioma cells migration. Collectively, our data show circSMARCA5 as a promising druggable tumor suppressor in GBM and suggest that it may exert its function by tethering the RBP SRSF1.

Data-assisted protein structure modeling by global optimization in CASP12.

In CASP12, 2 types of data-assisted protein structure modeling were experimented. Either SAXS experimental data or cross-linking experimental data was provided for a selected number of CASP12 targets that the CASP12 predictor could utilize for better protein structure modeling. We devised 2 separate energy terms for SAXS data and cross-linking data to drive the model structures into more native-like structures that satisfied the given experimental data as much as possible. In CASP11, we successfully performed protein structure modeling using simulated sparse and ambiguously assigned NOE data and/or correct residue-residue contact information, where the only energy term that folded the protein into its native structure was the term which was originated from the given experimental data. However, the 2 types of experimental data provided in CASP12 were far from being sufficient enough to fold the target protein into its native structure because SAXS data provides only the overall shape of the molecule and the cross-linking contact information provides only very low-resolution distance information. For this reason, we combined the SAXS or cross-linking energy term with our regular modeling energy function that includes both the template energy term and the de novo energy terms. By optimizing the newly formulated energy function, we obtained protein models that fit better with provided SAXS data than the X-ray structure of the target. However, the improvement of the model relative to the 1 modeled without the SAXS data, was not significant. Consistent structural improvement was achieved by incorporating cross-linking data into the protein structure modeling.

An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking.

Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and off-rates and crosslinking rate. From the on- and off-rate we obtain the occupancy and residence time. The following protocol is the second iteration of this method, CLKv2, updated with improved crosslinking and quenching conditions, more information about crosslinking rates, and systematic procedures for modeling the observed kinetic regimes. CLKv2 analysis has been applied to investigate the binding behavior of the TATA-binding protein (TBP), and a selected subset of other TFs. The protocol was developed using yeast cells, but may be applicable to cells from other organisms as well.

SF3B1 Gene Expression in Erythroid Cells Is Regulated By Intron Retention Via a Posttranscriptional Mechanism Involving Cryptic Exons Proposed to Function As Splicing Decoys

Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from erythroblasts treated with inhibitors of nonsense-mediated decay. That decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and decoy splice junctions were low in early stage erythroblasts and much higher in mature erythroblasts. Second, selected decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 3‘ splice site factors including U2AF1 and U2AF2 can bind specifically to 3‘ splice sites of intron 4's decoy exons. Finally, several experiments showed that IR-promoting activity of decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 decoy exons could promote IR in heterologous contexts, but also that predicted decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late erythroblasts are controlled by decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. DisclosuresNo relevant conflicts of interest to declare.

Probing the metastable state of influenza hemagglutinin

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.

Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σK

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction of Bacillus subtilis IP SpoIVFB with its substrate Pro-σK depends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-β-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σK Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB-Pro-σK complex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σK loops into the membrane domain of SpoIVFB, and the rest of Pro-σK interacts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σK cleavage by the membrane domain.

Chaperone‐client interactions between Hsp21 and client proteins monitored in solution by small angle X‐ray scattering and captured by crosslinking mass spectrometry

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N- and C-terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast-localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N-terminal arms located on the outside of the dodecamer and six inwardly-facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small-angle X-ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone-client complexes were monitored and the Rg -values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N-terminal arms of the Hsp21 dodecamer. No such increase in Rg -values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone-client interactions involve the N-terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N-terminal arms of Hsp21 can trap and refold early-unfolding intermediates with or without dodecamer dissociation.

Accommodating Protein Dynamics in the Modeling of Chemical Crosslinks

Chemical crosslinking can identify the neighborhood relationships between specific amino-acid residues in proteins. The interpretation of crosslinking data is typically performed using single, static atomic structures. However, proteins are dynamic, undergoing motions spanning from local fluctuations of individual residues to global motions of protein assemblies. Here we demonstrate that failure to explicitly accommodate dynamics when interpreting crosslinks structurally can lead to considerable errors. We present a method and associated software, DynamXL, which is able to account directly for flexibility in the context of crosslinking modeling. Our benchmarking on a large dataset of model structures demonstrates significantly improved rationalization of experimental crosslinking data, and enhanced performance in a protein-protein docking protocol. These advances will provide a considerable increase in the structural insights attainable using chemical crosslinking coupled to mass spectrometry.

Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation

ABSTRACTPtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.

Exhaustively Identifying Cross-Linked Peptides with a Linear Computational Complexity

Chemical cross-linking coupled to mass spectrometry is a powerful tool to study protein-protein interactions and protein conformations. Two linked peptides are ionized and fragmented to produce a tandem mass spectrum. In such an experiment, a tandem mass spectrum contains ions from two peptides. The peptide identification problem becomes a peptide-peptide pair identification problem. Currently, most tools do not search all possible pairs due to the quadratic time complexity. Consequently, missed findings are unavoidable. In our previous work, we developed a tool named ECL to search all pairs of peptides exhaustively. Unfortunately, it is very slow due to the quadratic computational complexity, especially when the database is large. Furthermore, ECL uses a score function without statistical calibration, while researchers1-3 have proposed that it is inappropriate to directly compare uncalibrated scores because different spectra have different random score distributions. Here we propose an advanced version of ECL, named ECL2. It achieves a linear time and space complexity by taking advantage of the additive property of a score function. It can search a data set containing tens of thousands of spectra against a database containing thousands of proteins in a few hours. Comparison with other five state-of-the-art tools shows that ECL2 is much faster than pLink, StavroX, ProteinProspector, and ECL. Kojak is the only one that is faster than ECL2, but Kojak does not exhaustively search all possible peptide pairs. The comparison shows that ECL2 has the highest sensitivity among the state-of-the-art tools. The experiment using a large-scale in vivo cross-linking data set demonstrates that ECL2 is the only tool that can find the peptide-spectrum matches (PSMs) passing the false discovery rate/q-value threshold. The result illustrates that the exhaustive search and a well-calibrated score function are useful to find PSMs from a huge search space.

To Be or Not to Be? Five Guidelines to Avoid Misassignments in Cross-Linking/Mass Spectrometry.

The number of publications in the field of chemical cross-linking/mass spectrometry (MS) for deriving protein 3D structures and for probing protein/protein interactions has largely increased during the last years. MS analysis of the large cross-linking data sets requires an automated data analysis by dedicated software tools, but applying scoring procedures with statistical methods does not eliminate the fundamental problems of a misassignment of cross-linked products. In fact, we have observed a significant rate of misassigned cross-links in a number of publications, mainly due to the presence of isobaric cross-linked species, an incomplete fragmentation of cross-linked products, and low-mass accuracy fragment ion data. These false assignments will eventually lead to wrong conclusions on the structural information derived from chemical cross-linking/MS experiments. In this contribution, we examine the most common sources for misassigning cross-linked products. We propose and discuss rational criteria and suggest five guidelines that might be followed for a reliable and unambiguous identification of cross-links, independent of the software used for data analysis. In the interest of the cross-linking/MS approach, it should be ensured that only high-quality data enter the structural biology literature. Clearly, there is an urgent need to define common standards for data analysis and reporting formats of cross-linked products.

OmiXcore: a web server for prediction of protein interactions with large RNA.

SummaryHere we introduce omiXcore, a server for calculations of protein binding to large RNAs (> 500 nucleotides). Our webserver allows (i) use of both protein and RNA sequences without size restriction, (ii) pre-compiled library for exploration of human long intergenic RNAs interactions and (iii) prediction of binding sites.Results omiXcore was trained and tested on enhanced UV Cross-Linking and ImmunoPrecipitation data. The method discriminates interacting and non-interacting protein-RNA pairs and identifies RNA binding sites with Areas under the ROC curve > 0.80, which suggests that the tool is particularly useful to prioritize candidates for further experimental validation.Availability and implementation omiXcore is freely accessed on the web at http://service.tartaglialab.com/grant_submission/omixcore.Supplementary information Supplementary data are available at Bioinformatics online.

Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex

The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and Rtf1, and binds to the RNA polymerase II (Pol II) transcription elongation complex (EC). Here we report the reconstitution of Paf1C from Saccharomyces cerevisiae, and a structural analysis of Paf1C bound to a Pol II EC containing the elongation factor TFIIS. Cryo-electron microscopy and crosslinking data reveal that Paf1C is highly mobile and extends over the outer Pol II surface from the Rpb2 to the Rpb3 subunit. The Paf1-Leo1 heterodimer and Cdc73 form opposite ends of Paf1C, whereas Ctr9 bridges between them. Consistent with the structural observations, the initiation factor TFIIF impairs Paf1C binding to Pol II, whereas the elongation factor TFIIS enhances it. We further show that Paf1C is globally required for normal mRNA transcription in yeast. These results provide a three-dimensional framework for further analysis of Paf1C function in transcription through chromatin.

Exploring contacts of eRF1 with the 3′-terminus of the P site tRNA and mRNA stop signal in the human ribosome at various translation termination steps

Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3′-terminus to investigate mutual arrangement of the main components of translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3′-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals.

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state.

Intron Retention Mechanisms That Regulate SF3B1 and Mitoferrin Gene Expression during Late Erythropoiesis

Intron retention (IR) regulates hundreds of erythroid genes in a differentiation stage-specific manner during terminal erythropoiesis. Regulated genes include highly expressed RNA binding proteins (RBPs) such as SF3B1, as well as iron transporters (e.g., mitoferrins 1 and 2), and cytoskeletal proteins (e.g., alpha spectrin). Selected IR transcripts are relatively abundant; 25-50% of the above-mentioned transcripts can be polyadenylated, retained in the nucleus, and efficiently spliced at all but one or two introns, thus limiting the amount of translatable cytoplasmic mRNA. We are studying novel IR regulatory mechanisms involving both splice site strength and deeper intronic elements, using model introns that are differentially regulated in human erythroblasts. SF3B1, a key pre-RNA splicing factor implicated in MDS, exhibits dynamic regulation of intron 4, with low IR in proerythroblasts and high IR in mature orthochromatic erythroblasts. Intron 4 sequences include three ultraconserved elements that encode cryptic exons we term 4a, 4b, and 4c, two of which (4a and 4c) encode premature termination codons (PTCs) expected to induce nonsense-mediated decay (NMD) if spliced into SF3B1 transcripts. We hypothesize that these PTC exons can have an additional function as inefficiently spliced decoy(s), that is, their splice sites may interact with splice sites at the boundaries of intron 4 to form non-productive complexes that do not permit efficient splicing but instead prevent excision of the intron. This concept represents an extension of decoy models previously proposed by others to explain selected exon skipping events, and is supported by several recent findings: eCLIP (enhanced cross-linking and immunoprecipitation) data indicate strong binding of 3' splice site factors U2AF1 and U2AF2 at these exons; low levels of exon 4a and 4c splicing can be seen in NMD-inhibited cells; and deletion of exon 4c from minigene splicing reporters decreases IR. Besides SF3B1, we identified numerous other dynamically-regulated IR events encompassing cryptic PTC exons that bind 3' splice site factors. In contrast to the dynamic regulation of IR in SF3B1, IR in SLC25A37 (mitoferrin-1) and SLC25A28 (mitoferrin-2) is stably maintained at a high level throughout terminal erythropoiesis. Baseline IR levels for stably retained introns correlate with splice site strength, but are also influenced by deeper intronic elements. For example, SLC25A28 intron 2 contains intronic elements that appear to reduce IR, since blocking them with antisense morpholinos leads to substantially increased IR levels. These studies demonstrate that IR in major erythroid genes is regulated by sequences within the retained introns that can either increase or decrease retention, suggesting that multiple IR pathways are employed during terminal erythropoiesis to regulate gene expression. DisclosuresNo relevant conflicts of interest to declare.