scholarly journals Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities

2015 ◽  
Vol 112 (34) ◽  
pp. E4726-E4734 ◽  
Author(s):  
Jian Zhang ◽  
Yen K. Lieu ◽  
Abdullah M. Ali ◽  
Alex Penson ◽  
Kathryn S. Reggio ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Splicing Factors ◽  
Binding Affinities ◽  
Protein Protein Interactions ◽  
Mrna Isoforms ◽  
Exon Inclusion ◽  
Wild Type Counterpart ◽  
Derivative Bound ◽  
Shed Light

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein–protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

scholarly journals Computational Prediction of Protein-Protein Interactions of Human Tyrosinase

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Su-Fang Wang ◽  
Sangho Oh ◽  
Yue-Xiu Si ◽  
Zhi-Jiang Wang ◽  
Hong-Yan Han ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
3D Structure ◽  
Computational Prediction ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
Lim Domains ◽  
Binding Partners ◽  
Computational Predictions ◽  
Human Tyrosinase

The various studies on tyrosinase have recently gained the attention of researchers due to their potential application values and the biological functions. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein-protein interactions between tyrosinase and three binding partners, four and half LIM domains 2 (FHL2), cytochrome b-245 alpha polypeptide (CYBA), and RNA-binding motif protein 9 (RBM9). Our interaction simulations showed significant binding energy scores of −595.3 kcal/mol for FHL2, −859.1 kcal/mol for CYBA, and −821.3 kcal/mol for RBM9. We also investigated the residues of each protein facing toward the predicted site of interaction with tyrosinase. Our computational predictions will be useful for elucidating the protein-protein interactions of tyrosinase and studying its binding mechanisms.

scholarly journals Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

2006 ◽  
Vol 188 (12) ◽  
pp. 4169-4182 ◽  
Author(s):  
Michael Y. Galperin
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Transcriptional Regulators ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Response Regulators ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Archaeal Species

ABSTRACT CheY-like phosphoacceptor (or receiver [REC]) domain is a common module in a variety of response regulators of the bacterial signal transduction systems. In this work, 4,610 response regulators, encoded in complete genomes of 200 bacterial and archaeal species, were identified and classified by their domain architectures. Previously uncharacterized output domains were analyzed and, in some cases, assigned to known domain families. Transcriptional regulators of the OmpR, NarL, and NtrC families were found to comprise almost 60% of all response regulators; transcriptional regulators with other DNA-binding domains (LytTR, AraC, Spo0A, Fis, YcbB, RpoE, and MerR) account for an additional 6%. The remaining one-third is represented by the stand-alone REC domain (∼14%) and its combinations with a variety of enzymatic (GGDEF, EAL, HD-GYP, CheB, CheC, PP2C, and HisK), RNA-binding (ANTAR and CsrA), protein- or ligand-binding (PAS, GAF, TPR, CAP_ED, and HPt) domains, or newly described domains of unknown function. The diversity of domain architectures and the abundance of alternative domain combinations suggest that fusions between the REC domain and various output domains is a widespread evolutionary mechanism that allows bacterial cells to regulate transcription, enzyme activity, and/or protein-protein interactions in response to environmental challenges. The complete list of response regulators encoded in each of the 200 analyzed genomes is available online at http://www.ncbi.nlm.nih.gov/Complete_Genomes/RRcensus.html .

scholarly journals Targeting RNA Binding Proteins Involved in Neurodegeneration

2013 ◽  
Vol 18 (9) ◽  
pp. 967-983 ◽  
Author(s):  
Maurizio Romano ◽  
Emanuele Buratti
Keyword(s):  
Protein Interactions ◽  
Rna Processing ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Correct Identification ◽  
Protein Protein Interactions ◽  
Aggregation Processes ◽  
Cellular Pathways ◽  
Screening Approaches

Dysfunctions at the level of RNA processing have recently been shown to play a fundamental role in the pathogenesis of many neurodegenerative diseases. Several proteins responsible for these dysfunctions (TDP-43, FUS/TLS, and hnRNP A/Bs) belong to the nuclear class of heterogeneous ribonucleoproteins (hnRNPs) that predominantly function as general regulators of both coding and noncoding RNA metabolism. The discovery of the importance of these factors in mediating neuronal death has represented a major paradigmatic shift in our understanding of neurodegenerative processes. As a result, these discoveries have also opened the way toward novel biomolecular screening approaches in our search for therapeutic options. One of the major hurdles in this search is represented by the correct identification of the most promising targets to be prioritized. These may include aberrant aggregation processes, protein-protein interactions, RNA-protein interactions, or specific cellular pathways altered by disease. In this review, we discuss these four major options together with their various advantages and drawbacks.

scholarly journals iSUMO - integrative prediction of functionally relevant SUMOylation events

2016 ◽  
Author(s):  
Xiaotong Yao ◽  
Shuvadeep Maity ◽  
Shashank Gandhi ◽  
Marcin Imielenski ◽  
Christine Vogel
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
False Positive Rate ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Positive Rate ◽  
Protein Nucleic Acid ◽  
Scale Experiment

AbstractPost-translational modifications by the Small Ubiquitin-like Modifier (SUMO) are essential for diverse cellular functions. Large-scale experiment and sequence-based predictions have identified thousands of SUMOylated proteins. However, the overlap between the datasets is small, suggesting many false positives with low functional relevance. Therefore, we integrated ~800 sequence features and protein characteristics such as cellular function and protein-protein interactions in a machine learning approach to score likely functional SUMOylation events (iSUMO). iSUMO is trained on a total of 24 large-scale datasets, and it predicts 2,291 and 706 SUMO targets in human and yeast, respectively. These estimates are five times higher than what existing sequence-based tools predict at the same 5% false positive rate. Protein-protein and protein-nucleic acid interactions are highly predictive of protein SUMOylation, supporting a role of the modification in protein complex formation. We note the marked prevalence of SUMOylation amongst RNA-binding proteins. We validate iSUMO predictions by experimental or other evidence. iSUMO therefore represents a comprehensive tool to identify high-confidence, functional SUMOylation events for human and yeast.

scholarly journals The structural evolution of host-pathogen protein interactions: an integrative approach

2019 ◽  
Author(s):  
Anderson F. Brito ◽  
John W. Pinney
Keyword(s):  
Protein Interactions ◽  
Homology Modelling ◽  
Structural Evolution ◽  
Integrative Approach ◽  
Binding Affinities ◽  
Protein Protein Interactions ◽  
Host Pathogen ◽  
Cell Membrane Proteins ◽  
Pathogen Protein ◽  
History Of

ABSTRACTThe evolution of protein-protein interactions (PPIs) is directly influenced by the evolutionary histories of the genes and the species encoding the interacting proteins. When it comes to PPIs of host-pathogen systems, the complexity of their evolution is much higher, as two independent, but biologically associated entities, are involved. In this work, an integrative approach combining phylogenetics, tree reconciliations, ancestral sequence reconstructions, and homology modelling is proposed for studying the evolution of host-pathogen PPIs. As a case study, we analysed the evolution of interactions between herpesviral glycoproteins gD/gG and the cell membrane proteins nectins. By modelling the structures of more than 12,000 ancestral states of these virus-host complexes it was found that in early times of their evolution, these proteins were unable to interact, most probably due to electrostatic incompatibilities between their interfaces. After the event of gene duplication that gave rise to a paralog of gD (known as gG), both protein lineages evolved following distinct functional constraints, with most gD reaching high binding affinities towards nectins, while gG lost such ability, most probably due to a process of neofunctionalization. Based on their favourable interaction energies (negative ΔG), it is possible to hypothesize that apart from nectins 1 and 2, some alphaherpesviruses might also use nectins 3 and 4 as cell receptors. These findings show that the proposed integrative method is suitable for modelling the evolution of host-pathogen protein interactions, and useful for raising new hypotheses that broaden our understanding about the evolutionary history of PPIs, and their molecular functioning.

scholarly journals Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

2020 ◽  
Author(s):  
Christopher R. Carlson ◽  
Jonathan B. Asfaha ◽  
Chloe M. Ghent ◽  
Conor J. Howard ◽  
Nairi Hartooni ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Viral Genome ◽  
Rna Binding ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
N Protein ◽  
Binding Domains ◽  
Genome Transcription ◽  
Liquid Liquid Phase Separation

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell1–3. The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder4. Phosphorylation of the central disordered region is required for normal viral genome transcription5,6, which occurs in a cytoplasmic structure called the replication transcription complex (RTC)7–11. It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates12–15. Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

scholarly journals Quantitative yeast-yeast two hybrid for discovery and binding affinity estimation of protein-protein interactions

2020 ◽  
Author(s):  
Kaitlyn Bacon ◽  
Abigail Blain ◽  
John Bowen ◽  
Matthew Burroughs ◽  
Nikki McArthur ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Protein Interactions ◽  
Quantitative Estimation ◽  
Surface Display ◽  
Combinatorial Libraries ◽  
Yeast Surface Display ◽  
Binding Affinities ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid

AbstractQuantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two hybrid (qYY2H) system that not only enables discovery of specific protein-protein interactions, but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semi-empirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of the apparent KD of yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

scholarly journals Imaging within single NPCs reveals NXF1’s role in mRNA export on the cytoplasmic side of the pore

2019 ◽  
Vol 218 (9) ◽  
pp. 2962-2981 ◽  
Author(s):  
Rakefet Ben-Yishay ◽  
Amir Mor ◽  
Amit Shraga ◽  
Asaf Ashkenazy-Titelman ◽  
Noa Kinor ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mrna Export ◽  
Living Cells ◽  
Nuclear Pore ◽  
Protein Protein Interactions ◽  
Superresolution Microscopy ◽  
Directional Flow ◽  
Cytoplasmic Side

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein–protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

scholarly journals UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

2016 ◽  
Vol 72 (4) ◽  
pp. 497-511 ◽  
Author(s):  
Galina A. Stepanyuk ◽  
Pedro Serrano ◽  
Eigen Peralta ◽  
Carol L. Farr ◽  
Herbert L. Axelrod ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Splicing Factor ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Protein Protein Interactions ◽  
Rrm Domain ◽  
Nmr Structures ◽  
Binding Interface ◽  
Cell Extracts

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

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