Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation.

UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2's third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B's disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3's middle-domain elucidates its essential role in NMD.

Blasticidin S inhibits mammalian translation and enhances production of protein encoded by nonsense mRNA.

Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility.

Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches-including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering-we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion.

The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1.

Y-family DNA polymerases, such as polymerase η, polymerase ι, and polymerase κ, catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein Rev1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved Rev1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of Rev1. Yeast polymerase η, a prototypical translesion synthesis polymerase, binds both PCNA and Rev1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase η mediates its interactions both with PCNA and with Rev1. Moreover, the PIP motif of polymerase η binds in the hydrophobic pocket on the Rev1 C-terminal domain. We also show that the RIR motif of human polymerase κ and the PIP motif of yeast Msh6 bind both PCNA and Rev1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and Rev1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed.

Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures.

A balance of van der Waals, electrostatic, and hydrophobic forces drive the folding and packing of protein side chains. Although such interactions between residues are often approximated as being pairwise additive, in reality, higher-order many-body contributions that depend on environment drive hydrophobic collapse and cooperative electrostatics. Beginning from dead-end elimination, we derive the first algorithm, to our knowledge, capable of deterministic global repacking of side chains compatible with many-body energy functions. The approach is applied to seven PCNA x-ray crystallographic data sets with resolutions 2.5-3.8 Å (mean 3.0 Å) using an open-source software. While PDB_REDO models average an Rfree value of 29.5% and MOLPROBITY score of 2.71 Å (77th percentile), dead-end elimination with the polarizable AMOEBA force field lowered Rfree by 2.8-26.7% and improved mean MOLPROBITY score to atomic resolution at 1.25 Å (100th percentile). For structural biology applications that depend on side-chain repacking, including x-ray refinement, homology modeling, and protein design, the accuracy limitations of pairwise additivity can now be eliminated via polarizable or quantum mechanical potentials.

New insights into no-go, non-stop and nonsense-mediated mRNA decay complexes.

Eukaryotes possess a variety of translational control mechanisms which function in the surveillance of mRNAs, discriminating between normal and aberrant translation elongation and termination, triggering mRNA decay. The three major evolutionarily conserved eukaryotic pathways are No-Go, Non-Stop and Nonsense-Mediated mRNA Decay. Recent findings suggest that stalling of the ribosome, due to mRNA secondary structure or translation into poly(A)-stretches, leads to ribosome collisions which are detected by No-Go/Non-Stop mRNA decay factors. Subsequent ribosome ubiquitination at the interface of two collided ribosomes is considered the signal for mRNA decay. Similarly, translation termination at a premature stop codon is slower than normal, leading to recruitment and activation of nonsense-mediated mRNA decay factors, including SMG1-8-9. Here, we detail new insights into the molecular mechanisms of these pathways.

Modeling Conformationally Flexible Proteins With X-ray Scattering and Molecular Simulations.

Proteins and protein complexes with high conformational flexibility participate in a wide range of biological processes. These processes include genome maintenance, gene expression, signal transduction, cell cycle regulation, and many others. Gaining a structural understanding of conformationally flexible proteins and protein complexes is arguably the greatest problem facing structural biologists today. Over the last decade, some progress has been made toward understanding the conformational flexibility of such systems using hybrid approaches. One particularly fruitful strategy has been the combination of small-angle X-ray scattering (SAXS) and molecular simulations. In this article, we provide a brief overview of SAXS and molecular simulations and then discuss two general approaches for combining SAXS data and molecular simulations: minimal ensemble approaches and full ensemble approaches. In minimal ensemble approaches, one selects a minimal ensemble of structures from the simulations that best fit the SAXS data. In full ensemble approaches, one validates a full ensemble of structures from the simulations using SAXS data. We argue that full ensemble models are more realistic than minimal ensemble searches models and that full ensemble approaches should be used wherever possible.

Eukaryotic translesion synthesis: Choosing the right tool for the job.

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases are thought to form a complex at the stalled replication fork that we refer to as the mutasome. This hypothetical multi-protein complex is structurally organized by the replication accessory factor PCNA and the non-classical polymerase Rev1. One of the non-classical polymerases within this complex then catalyzes replication through the damage. Each non-classical polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical polymerase is chosen to bypass the damage. In this review article, we discuss how the most appropriate polymerase is chosen. In so doing, we examine the structural motifs that mediate the protein interactions in the mutasome; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome.

Conformational Flexibility of Ubiquitin-Modified and SUMO-Modified PCNA Shown by Full-Ensemble Hybrid Methods.

Ubiquitin-modified proliferating cell nuclear antigen (PCNA) and small ubiquitin-like modifier (SUMO)-modified PCNA regulate DNA damage tolerance pathways. X-ray crystal structures of these proteins suggested that they do not have much conformational flexibility because the modifiers have preferred binding sites on the surface of PCNA. By contrast, small-angle X-ray scattering analyses of these proteins suggested that they have different degrees of conformational flexibility, with SUMO-modified PCNA being more flexible. These conclusions were based on minimal-ensemble hybrid approaches, which produce unrealistic models by representing flexible proteins with only a few static structures. To overcome the limitations of minimal-ensemble hybrid approaches and to determine the degree of conformational flexibility of ubiquitin-modified PCNA and SUMO-modified PCNA, we utilized a novel full-ensemble hybrid approach. We carried out molecular simulations and small-angle X-ray scattering analyses of both proteins and obtained outstanding agreement between the full ensembles generated by the simulations and the experimental data. We found that both proteins have a high degree of conformational flexibility. The modifiers occupy many positions around the back and side of the PCNA ring. Moreover, we found no preferred ubiquitin-binding or SUMO-binding sites on PCNA. This conformational flexibility likely facilitates the recognition of downstream effector proteins and the formation of PCNA tool belts.

Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases.

Translesion synthesis is the process by which nonclassical DNA polymerases bypass DNA damage during DNA replication. Cells possess a variety of nonclassical polymerases, each one is specific for incorporating nucleotides opposite to one or more closely related DNA lesions, called its cognate lesions. In this chapter, we discuss a variety of approaches for probing the catalytic activities and the protein-protein interactions of nonclassical polymerases. With respect to their catalytic activities, we discuss polymerase assays, steady-state kinetics, and presteady-state kinetics. With respect to their interactions, we discuss qualitative binding assays such as enzyme-linked immunosorbent assays and coimmunoprecipitation; quantitative binding assays such as isothermal titration calorimetry, surface plasmon resonance, and nuclear magnetic resonance spectroscopy; and single-molecule binding assays such as total internal reflection fluorescence microscopy. We focus on how nonclassical polymerases accommodate their cognate lesions during nucleotide incorporation and how the most appropriate nonclassical polymerase is selected for bypassing a given lesion.

Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.