Enhanced immunoprecipitation techniques for the identification of RNA-binding protein partners: IGF2BP1 interactions in mammary epithelial cells

RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested “exclusion-based” purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP–RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.     

动植物中RNA结合蛋白的研究进展

RNA结合蛋白(RNA-binding proteins)在转录后基因表达调节中起着重要的作用,它通过和RNA相互作用来调节细胞的功能。RNA结合蛋白参与RNA剪接、多聚腺苷化作用、序列编辑、RNA转运、维持RNA的稳定和降解、细胞内定位和翻译控制等RNA代谢的各个方面。主要介绍了RNA结合蛋白的结构、靶标RNA及RNA结合蛋白在动植物和疾病中的研究。     

RNA-binding proteins in neurological diseases

Emerging studies support that RNA-binding proteins(RBPs)play critical roles in human biology and pathogenesis.RBPs are essential players in RNA processing and metabolism,including pre-mRNA splicing,polyadenylation,transport,surveillance,mRNA localization,mRNA stability control,translational control and editing of various types of RNAs.Aberrant expression of and mutations in RBP genes affect various steps of RNA processing,altering target gene function.RBPs have been associated with various diseases,including neurological diseases.Here,we mainly focus on selected RNA-binding proteins including Nova-1/Nova-2,HuR/HuB/HuC/HuD,TDP-43,Fus,Rbfox1/Rbfox2,QKI and FMRP,discussing their function and roles in human diseases.     

CeLINC,a fluorescence-based protein–protein interaction assay in Caenorhabditis elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein–protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein–protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.     

Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms

Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5′-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.     

Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform

Caenorhabditis elegans GLD-3 is a five K homology (KH) domain-containing protein involved in the translational control of germline-specific mRNAs during embryogenesis. GLD-3 interacts with the cytoplasmic poly(A)-polymerase GLD-2. The two proteins cooperate to recognize target mRNAs and convert them into a polyadenylated, translationally active state. We report the 2.8-Å-resolution crystal structure of a proteolytically stable fragment encompassing the KH2, KH3, KH4, and KH5 domains of C. elegans GLD-3. The structure reveals that the four tandem KH domains are organized into a globular structural unit. The domains are involved in extensive side-by-side interactions, similar to those observed in previous structures of dimeric KH domains, as well as head-to-toe interactions. Small-angle X-ray scattering reconstructions show that the N-terminal KH domain (KH1) forms a thumb-like protrusion on the KH2–KH5 unit. Although KH domains are putative RNA-binding modules, the KH region of GLD-3 is unable in isolation to cross-link RNA. Instead, the KH1 domain mediates the direct interaction with the poly(A)-polymerase GLD-2, pointing to a function of the KH region as a protein–protein interaction platform.     

Match_Motif: A rapid computational tool to assist in protein–protein interaction design

In order to generate protein assemblies with a desired function, the rational design of protein–protein binding interfaces is of significant interest. Approaches based on random mutagenesis or directed evolution may involve complex experimental selection procedures. Also, molecular modeling approaches to design entirely new proteins and interactions with partner molecules can involve large computational efforts and screening steps. In order to simplify at least the initial effort for designing a putative binding interface between two proteins the Match_Motif approach has been developed. It employs the large collection of known protein–protein complex structures to suggest interface modifications that may lead to improved binding for a desired input interaction geometry. The approach extracts interaction motifs based on the backbone structure of short (four residues) segments and the relative arrangement with respect to short segments on the partner protein. The interaction geometry is used to search through a database of such motifs in known stable bound complexes. All matches are rapidly identified (within a few seconds) and collected and can be used to guide changes in the interface that may lead to improved binding. In the output, an alternative interface structure is also proposed based on the frequency of occurrence of side chains at a given interface position in all matches and based on sterical considerations. Applications of the procedure to known complex structures and alternative arrangements are presented and discussed. The program, data files, and example applications can be downloaded from https://www.groups.ph.tum.de/t38/downloads/.     

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

It is recognized now that intrinsically disordered proteins (IDPs), which do not have unique 3D structures as a whole or in noticeable parts, constitute a significant fraction of any given proteome. IDPs are characterized by an astonishing structural and functional diversity that defines their ability to be universal regulators of various cellular pathways. Programmed cell death (PCD) is one of the most intricate cellular processes where the cell uses specialized cellular machinery and intracellular programs to kill itself. This cell-suicide mechanism enables metazoans to control cell numbers and to eliminate cells that threaten the animal''s survival. PCD includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). These modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein–protein interactions. To understand the role of the intrinsic disorder in controlling and regulating the PCD, several large sets of PCD-related proteins across 28 species were analyzed using a wide array of modern bioinformatics tools. This study indicates that the intrinsic disorder phenomenon has to be taken into consideration to generate a complete picture of the interconnected processes, pathways, and modules that determine the essence of the PCD. We demonstrate that proteins involved in regulation and execution of PCD possess substantial amount of intrinsic disorder. We annotate functional roles of disorder across and within apoptosis, autophagy, and necroptosis processes. Disordered regions are shown to be implemented in a number of crucial functions, such as protein–protein interactions, interactions with other partners including nucleic acids and other ligands, are enriched in post-translational modification sites, and are characterized by specific evolutionary patterns. We mapped the disorder into an integrated network of PCD pathways and into the interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathway.     

Accuracy modulating mutations of the ribosomal protein S4-S5 interface do not necessarily destabilize the rps4-rps5 protein–protein interaction

During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon–anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent crystallographic structures of the ribosome suggest that cognate tRNAs induce a “closed conformation” of the small subunit that stabilizes the codon–anticodon interactions at the A site. During formation of the closed conformation, the protein interface between rpS4 and rpS5 is broken while new contacts form with rpS12. Mutations in rpS12 confer streptomycin resistance or dependence and show a hyperaccurate phenotype. Mutations reversing streptomycin dependence affect rpS4 and rpS5. The canonical rpS4 and rpS5 streptomycin independent mutations increase translational errors and were called ribosomal ambiguity mutations (ram). The mutations in these proteins are proposed to affect formation of the closed complex by breaking the rpS4-rpS5 interface, which reduces the cost of domain closure and thus increases translational errors. We used a yeast two-hybrid system to study the interactions between the small subunit ribosomal proteins rpS4 and rpS5 and to test the effect of ram mutations on the stability of the interface. We found no correlation between ram phenotype and disruption of the interface.     

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

Signaling of semaphorin ligands via their plexin–neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.     

Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA-protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.     

HydRA: Deep-learning models for predicting RNA-binding capacity from protein interaction association context and protein sequence

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Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70’s EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.     

Structure of the Arabidopsis thaliana DCL4 DUF283 domain reveals a noncanonical double-stranded RNA-binding fold for protein–protein interaction

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an α-β-β-β-α topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal α helix of DUF283 runs cross over the C-terminal α helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.