GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression.

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5' cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP-GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.

Coupling 40S ribosome recruitment to modification of a cap-binding initiation factor by eIF3 subunit e.

40S ribosomes are loaded onto capped mRNAs via the multisubunit translation initiation factors eIF3 and eIF4F. While eIF4E is the eIF4F cap recognition component, the eIF4G subunit associates with 40S-bound eIF3. How this intricate process is coordinated remains poorly understood. Here, we identify an eIF3 subunit that regulates eIF4F modification and show that eIF3e is required for inducible eIF4E phosphorylation. Significantly, recruitment of the eIF4E kinase Mnk1 (MAPK signal-integrating kinase 1) to eIF4F depended on eIF3e, and eIF3e was sufficient to promote Mnk1-binding to eIF4G. This establishes a mechanism by which 40S ribosome loading imparts a phosphorylation mark on the cap-binding eIF4F complex that regulates selective mRNA translation and is synchronized by a specific eIF3 subunit.

The Leishmania PABP1-eIF4E4 interface: a novel 5'-3' interaction architecture for trans-spliced mRNAs.

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

Changes in conformational equilibria regulate the activity of the Dcp2 decapping enzyme.

Crystal structures of enzymes are indispensable to understanding their mechanisms on a molecular level. It, however, remains challenging to determine which structures are adopted in solution, especially for dynamic complexes. Here, we study the bilobed decapping enzyme Dcp2 that removes the 5' cap structure from eukaryotic mRNA and thereby efficiently terminates gene expression. The numerous Dcp2 structures can be grouped into six states where the domain orientation between the catalytic and regulatory domains significantly differs. Despite this wealth of structural information it is not possible to correlate these states with the catalytic cycle or the activity of the enzyme. Using methyl transverse relaxation-optimized NMR spectroscopy, we demonstrate that only three of the six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalytically active state. We show how mRNA substrate and the activator proteins Dcp1 and Edc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly, the active state of the complex is only stably formed in the presence of both activators and the mRNA substrate or the m7GDP decapping product, which we rationalize based on a crystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA.

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins.

The mRNA 5' cap consists of N7-methylguanosine bound by a 5',5'-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap-protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap-protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

Molecular recognition of mRNA 5' cap by 3' poly(A)-specific ribonuclease (PARN) differs from interactions known for other cap-binding proteins.

The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action. We used surface plasmon resonance kinetic analysis, quantitative equilibrium fluorescence titrations and circular dichroism to study the cap binding properties of PARN. The molecular mechanism of 5' cap recognition by PARN has been demonstrated to differ from interactions seen for other known cap-binding proteins in that: i) the auxiliary biological function of 5' cap binding by the 3' degrading enzyme is accomplished by negative cooperativity of PARN dimer subunits; ii) non-coulombic interactions are major factors in the complex formation; and iii) PARN has versatile activity toward alternative forms of the cap. These characteristics contribute to stabilization of the PARN-cap complex needed for the deadenylation processivity. Our studies provide a consistent biophysical basis for elucidation of the processive mechanism of PARN-mediated 3' mRNA deadenylation and provide a new framework to interpret the role of the 5' cap in mRNA degradation.

A molecular modelling approach to understand the effect of co-evolutionary mutations (V344M, I354L) identified in the PB2 subunit of influenza A 2009 pandemic H1N1 virus on m7GTP ligand binding.

The cap binding domain of the polymerase basic 2 (PB2) subunit of influenza polymerases plays a critical role in mediating the 'cap-snatching' mechanism by binding the 5' cap of host pre-mRNAs during viral mRNA transcription. Monitoring variations in the PB2 protein is thus vital for evaluating the pathogenic potential of the virus. Based on selection pressure analysis of PB2 gene sequences of the pandemic H1N1 (pH1N1) viruses of the period 2009-2014, we identified a site, 344V/M, in the vicinity of the cap binding pocket showing evidence of adaptive evolution and another co-evolving residue, 354I/L, in close vicinity. Modelling of the three-dimensional structure of the pH1N1 PB2 cap binding domain, docking of the pre-mRNA cap analogue m7GTP and molecular dynamics simulation studies of the docked complexes performed for four PB2 variants observed showed that the complex possessing V344M with I354L possessed better ligand binding affinity due to additional hydrogen bond contacts between m7GTP and the key residues His432 and Arg355 that was attributed to a displacement of the 424 loop and a flip of the side chain of Arg355, respectively. The co-evolutionary mutations identified (V344M, I354L) were found to be established in the PB2 gene of the pH1N1 viral population over the period 2010-2014. The study demonstrates the molecular basis for the enhanced m7GTP ligand binding affinity with the 344M-354L synergistic combination in PB2. Furthermore, the insight gained into understanding the molecular mechanism of cap binding in pH1N1 viruses may be useful for designing novel drugs targeting the PB2 cap binding domain.

Cap-binding complex (CBC).

The 7mG (7-methylguanosine cap) formed on mRNA is fundamental to eukaryotic gene expression. Protein complexes recruited to 7mG mediate key processing events throughout the lifetime of the transcript. One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation. Gene expression can be regulated by signalling pathways which influence CBC function. The aim of the present review is to discuss the mechanisms by which CBC mediates and co-ordinates multiple gene expression events.

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.

Cap-binding proteins have been routinely isolated using m7GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m7GTP. Here, we report the synthesis of new affinity resins, m7GpCH2pp- and m7GpCH2ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m7GpppA-Sepharose, bind recombinant mouse eIF4E²8⁻²¹7 specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m7GpCH2pp- and m7GpCH2ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m7GpCH2ppA-Sepharose. Our data prove the applicability of these novel resins, especially m7GpCH2ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.

Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1-7-Pat1 complex.

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa- or heptameric RNA-binding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 5'-3' mRNA decay pathway. This complex also protects mRNA 3'-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 3'-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 3'-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.

Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition.

Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the K(M) for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 proline-rich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2.

eIF4E-homologous protein (4EHP): a multifarious cap-binding protein.

The cap-binding protein 4EHP/eIF4E2 has been a recent object of interest in the field of post-transcriptional gene regulation and translational control. From ribosome-associated quality control, to RNA decay and microRNA-mediated gene silencing, this member of the eIF4E protein family regulates gene expression through numerous pathways. Low in abundance but ubiquitously expressed, 4EHP interacts with different binding partners to form multiple protein complexes that regulate translation in a variety of biological contexts. Documented functions of 4EHP primarily relate to its role as a translational repressor, but recent findings indicate that it might also participate in the activation of translation in specific settings. In this review, we discuss the known functions, properties and mechanisms that involve 4EHP in the control of gene expression. We also discuss our current understanding of how 4EHP processes are regulated in eukaryotic cells, and the diseases implicated with dysregulation of 4EHP-mediated translational control.

The importin ß binding domain as a master regulator of nucleocytoplasmic transport.

Specific and efficient recognition of import cargoes is essential to ensure nucleocytoplasmic transport. To this end, the prototypical karyopherin importin ß associates with import cargoes directly or, more commonly, through import adaptors, such as importin α and snurportin. Adaptor proteins bind the nuclear localization sequence (NLS) of import cargoes while recruiting importin ß via an N-terminal importin ß binding (IBB) domain. The use of adaptors greatly expands and amplifies the repertoire of cellular cargoes that importin ß can efficiently import into the cell nucleus and allows for fine regulation of nuclear import. Accordingly, the IBB domain is a dedicated NLS, unique to adaptor proteins that functions as a molecular liaison between importin ß and import cargoes. This review provides an overview of the molecular role played by the IBB domain in orchestrating nucleocytoplasmic transport. Recent work has determined that the IBB domain has specialized functions at every step of the import and export pathway. Unexpectedly, this stretch of ~40 amino acids plays an essential role in regulating processes such as formation of the import complex, docking and translocation through the nuclear pore complex (NPC), release of import cargoes into the cell nucleus and finally recycling of import adaptors and importin ß into the cytoplasm. Thus, the IBB domain is a master regulator of nucleocytoplasmic transport, whose complex molecular function is only recently beginning to emerge. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry.

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m(3)G cap and snurportin 1. We were able to define previously unknown sites of protein-protein and protein-RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m(3)G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m(3)G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA.

Synthesis of a new class of ribose functionalized dinucleotide cap analogues for biophysical studies on interaction of cap-binding proteins with the 5' end of mRNA.

mRNAs of primitive eukaryotes such as Caenorhabditis elegans and Ascaris summ possess two different caps at their 5' terminus. They have either a typical cap which consists of 7-methylguanosine linked via a 5',5'-triphosphate bridge to the first transcribed nucleotide (MMG cap) or an atypical hypermethylated form with two additional methyl groups at the N2 position (TMG cap). Studies on interaction between the 5' end of mRNA and proteins that specifically recognize its structure have been carried out for several years and they often require chemically modified cap analogues. Here, we present the synthesis of five novel dinucleotide MMG and TMG cap analogues designed for binding studies using biophysical methods such as electron spin resonance (ESR) and surface plasmon resonance (SPR). New analogues were prepared by derivatization of the 2',3'-cis diol of the second nucleotide in the cap structure with levulinic acid, and coupling of the obtained acetal through its carboxylic group with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino TEMPO), ethylenediamine (EDA) or (+)-biotinyl-3,6,9-trioxaundecanediamine (amine-PEO(3)-biotin).

Polymerase translocation with respect to single-stranded nucleic acid: looping or wrapping of primer around a poly(A) polymerase.

Vaccinia virus protein VP55 translocates continuously with respect to single-stranded nucleic acid while extending its 3'end. Here, all key sites of polymerase-primer interaction were identified, demonstrating the wrapping or looping of polyadenylation primer around the polymerase during translocation. Side-chain substitutions at one of the sites indicated its requirement for tail extension beyond approximately 12 nucleotides in length, and conformational changes observed upon oligonucleotide binding suggested allosteric connectivity during translocation. Conformational changes in VP39 upon VP55 binding suggested that, within the VP55-VP39 complex, VP39's mRNA 5' cap binding site closes. The crystallographic structure showed a PAPase catalytic center without side-chain substitutions, possessing two metal ions and with all known reactive and catalytic groups represented, fitting a classical two-metal ion mechanism for phosphoryl transfer.

Conformational selection in the recognition of the snurportin importin beta binding domain by importin beta.

The structural flexibility of beta-karyopherins is critical to mediate the interaction with transport substrates, nucleoporins, and the GTPase Ran. In this paper, we provide structural evidence that the molecular recognition of the transport adaptor snurportin by importin beta follows the population selection mechanism. We have captured two drastically different conformations of importin beta bound to the snurportin importin beta binding domain trapped in the same crystallographic asymmetric unit. We propose the population selection may be a general mechanism used by beta-karyopherins to recognize transport substrates.

Molecular determinants of snurportin 1 ligand affinity and structural response upon binding.

The transport of large biomolecules such as proteins and RNA across nuclear pore complexes is a field of strong interest and research. Although the basic mechanisms are fairly well understood, the details of the underlying intermolecular interaction within these transport complexes are still unclear. The recognition dynamics and energetics of cargo binding to the transport receptor are not yet resolved. Here, the binding of dimethylated RNA-caps to snurportin 1 is studied by molecular-dynamics simulations. The simulations reveal a strong structural response of the protein upon RNA-cap release. In particular, major rearrangements occur in regions already intrinsically flexible in the holo structure. Additionally, the difference in free energy of binding to snurportin 1 between the two methylation states of the RNA-cap, responsible for the directionality of the transport is quantified. In particular, desolvation of the ligand is revealed as the key-step in binding to snurportin 1. These findings suggest that the binding of m(3)G-capped RNA is mainly driven by the enhanced water entropy gain of the solvation shell.

Accuracy and convergence of free energy differences calculated from nonequilibrium switching processes.

The molecular-dynamics-based calculation of accurate free energy differences for biomolecular systems is a challenging task. Accordingly, convergence and accuracy of established equilibrium methods has been subject of many studies, often focusing at small test systems. In contrast, the potential of more recently proposed nonequilibrium methods, derived from the Jarzynski and Crooks equalities, has not yet fully been explored. Here, we compare the performance of these methods by calculating free energy differences for test systems at different levels of complexity and varying extent of the involved perturbations. We consider the interconversion of ethane into methanol, the switching of a tryptophane-sidechain in a tripeptide, and the binding of two different ligands to the globular protein snurportin 1. On the basis of our results, we suggest and assess a new nonequilibrium free energy method, Crooks Gaussian Intersection (CGI), which combines the advantages of existing methods. CGI is highly parallelizable and, for the test systems considered here, is shown to outperform the other studied equilibrium and nonequilibrium methods.