Structural mechanism of angiogenin activation by the ribosome.

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.

Extended stop codon context predicts nonsense codon readthrough efficiency in human cells.

Protein synthesis terminates when a stop codon enters the ribosome's A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3'-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient's likelihood of improvement in response to drugs given their nonsense mutation sequence context.

Macroscopic Helical Assembly of One-Dimensional Coordination Polymers: Helicity Inversion Triggered by Solvent Isomerism.

Assembling macroscopic helices with controllable chirality and understanding their formation mechanism are highly desirable but challenging tasks for artificial systems, especially coordination polymers. Here, we utilize solvents as an effective tool to induce the formation of macroscopic helices of chiral coordination polymers (CPs) and manipulate their helical sense. We chose the Ni/R-,S-BrpempH2 system with a one-dimensional tubular structure, where R-,S-BrpempH2 stands for R-,S-(1-(4-bromophenyl)ethylaminomethylphosphonic acid). The morphology of the self-assemblies can be controlled by varying the cosolvent in water, resulting in the formation of twisted ribbons of R-,S-Ni(Brpemp)(H2O)·H2O (R-,S-2T) in pure H2O; needle-like crystals of R-,S-Ni(Brpemp)(H2O)2·1/3CH3CN (R-,S-1C) in 20 vol % CH3CN/H2O; nanofibers of R-,S-Ni(Brpemp)(H2O)·H2O (R-,S-3F) in 20-40 vol % methanol/H2O or ethanol/H2O; and superhelices of R-,S-Ni(Brpemp)(H2O)·H2O (R-,S-4H or 5H) in 40 vol % propanol/H2O. Interestingly, the helicity of the superhelix can be controlled by using a propanol isomer in water. For the Ni/R-BrpempH2 system, a left-handed superhelix of R-4H(M) was obtained in 40 vol % NPA/H2O, while a right-handed superhelix of R-5H(P) was isolated in 40 vol % IPA/H2O. These results were rationalized by theoretical calculations. Adsorption studies revealed the chiral recognition behavior of these compounds. This work may contribute to the development of chiral CPs with a macroscopic helical morphology and interesting functionalities.

Direct and indirect consequences of PAB1 deletion in the regulation of translation initiation, translation termination, and mRNA decay.

Cytoplasmic poly(A)-binding protein (PABPC; Pab1 in yeast) is thought to be involved in multiple steps of post-transcriptional control, including translation initiation, translation termination, and mRNA decay. To understand these roles of PABPC in more detail for endogenous mRNAs, and to distinguish its direct effects from indirect effects, we have employed RNA-Seq and Ribo-Seq to analyze changes in the abundance and translation of the yeast transcriptome, as well as mass spectrometry to assess the abundance of the components of the yeast proteome, in cells lacking the PAB1 gene. We observed drastic changes in the transcriptome and proteome, as well as defects in translation initiation and termination, in pab1Δ cells. Defects in translation initiation and the stabilization of specific classes of mRNAs in pab1Δ cells appear to be partly indirect consequences of reduced levels of specific initiation factors, decapping activators, and components of the deadenylation complex in addition to the general loss of Pab1's direct role in these processes. Cells devoid of Pab1 also manifested a nonsense codon readthrough phenotype indicative of a defect in translation termination, but this defect may be a direct effect of the loss of Pab1 as it could not be attributed to significant reductions in the levels of release factors.

Eukaryotic mRNA decapping factors: molecular mechanisms and activity.

Decapping is the enzymatic removal of 5' cap structures from mRNAs in eukaryotic cells. Cap structures normally enhance mRNA translation and stability, and their excision commits an mRNA to complete 5'-3' exoribonucleolytic digestion and generally ends the physical and functional cellular presence of the mRNA. Decapping plays a pivotal role in eukaryotic cytoplasmic mRNA turnover and is a critical and highly regulated event in multiple 5'-3' mRNA decay pathways, including general 5'-3' decay, nonsense-mediated mRNA decay (NMD), AU-rich element-mediated mRNA decay, microRNA-mediated gene silencing, and targeted transcript-specific mRNA decay. In the yeast Saccharomyces cerevisiae, mRNA decapping is carried out by a single Dcp1-Dcp2 decapping enzyme in concert with the accessory activities of specific regulators commonly known as decapping activators or enhancers. These regulatory proteins include the general decapping activators Edc1, 2, and 3, Dhh1, Scd6, Pat1, and the Lsm1-7 complex, as well as the NMD-specific factors, Upf1, 2, and 3. Here, we focus on in vivo mRNA decapping regulation in yeast. We summarize recently uncovered molecular mechanisms that control selective targeting of the yeast decapping enzyme and discuss new roles for specific decapping activators in controlling decapping enzyme targeting, assembly of target-specific decapping complexes, and the monitoring of mRNA translation. Further, we discuss the kinetic contribution of mRNA decapping for overall decay of different substrate mRNAs and highlight experimental evidence pointing to the functional coordination and physical coupling between events in mRNA deadenylation, decapping, and 5'-3' exoribonucleolytic decay.

Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance.

Upf1, Upf2, and Upf3, the central regulators of nonsense-mediated mRNA decay (NMD), appear to exercise their NMD functions while bound to elongating ribosomes, and evidence for this conclusion is particularly compelling for Upf1. Hence, we used selective profiling of yeast Upf1:ribosome association to define that step in greater detail, understand whether the nature of the mRNA being translated influences Upf1:80S interaction, and elucidate the functions of ribosome-associated Upf1. Our approach has allowed us to clarify the timing and specificity of Upf1 association with translating ribosomes, obtain evidence for a Upf1 mRNA surveillance function that precedes the activation of NMD, identify a unique ribosome state that generates 37-43 nt ribosome footprints whose accumulation is dependent on Upf1's ATPase activity, and demonstrate that a mutated form of Upf1 can interfere with normal translation termination and ribosome release. In addition, our results strongly support the existence of at least two distinct functional Upf1 complexes in the NMD pathway.

Functional restoration of mouse Nf1 nonsense alleles in differentiated cultured neurons.

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant genetic disorders, is caused by mutations in the NF1 gene. NF1 patients have a wide variety of manifestations with a subset at high risk for the development of tumors in the central nervous system (CNS). Nonsense mutations that result in the synthesis of truncated NF1 protein (neurofibromin) are strongly associated with CNS tumors. Therapeutic nonsense suppression with small molecule drugs is a potentially powerful approach to restore the expression of genes harboring nonsense mutations. Ataluren is one such drug that has been shown to restore full-length functional protein in several models of nonsense mutation diseases, as well as in patients with nonsense mutation Duchenne muscular dystrophy. To test ataluren's potential applicability to NF1 nonsense mutations associated with CNS tumors, we generated a homozygous Nf1R683X/R683X-3X-FLAG mouse embryonic stem (mES) cell line which recapitulates an NF1 patient nonsense mutation (c.2041 C > T; p.Arg681X). We differentiated Nf1R683X/R683X-3X-FLAG mES cells into cortical neurons in vitro, treated the cells with ataluren, and demonstrated that ataluren can promote readthrough of the nonsense mutation at codon 683 of Nf1 mRNA in neural cells. The resulting full-length protein is able to reduce the cellular level of hyperactive phosphorylated ERK (pERK), a RAS effector normally suppressed by the NF1 protein.

Dcp2 C-terminal cis-binding elements control selective targeting of the decapping enzyme by forming distinct decapping complexes.

A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5' to 3' decay, but the molecular mechanisms controlling mRNA selectivity by the enzyme remain elusive. Through extensive genetic analyses we reveal that Dcp2 C-terminal domain cis-regulatory elements control decapping enzyme target specificity by orchestrating formation of distinct decapping complexes. Two Upf1-binding motifs direct the decapping enzyme to nonsense-mediated mRNA decay substrates, a single Edc3-binding motif targets both Edc3 and Dhh1 substrates, and Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Scd6 and Xrn1 also have specific binding sites on Dcp2, allowing them to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, Scd6, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its substrate specificity and enzymatic activation.

Layer or Tube? Uncovering Key Factors Determining the Rolling-up of Layered Coordination Polymers.

Nanotubular materials have garnered considerable attention since the discovery of carbon nanotubes. Although the layer-to-tube rolling up mechanism has been well recognized in explaining the formation of many inorganic nanotubes, it has not been generally applied to coordination polymers (CPs). To uncover the key factors that determine the rolling-up of layered CPs, we have chosen the Co/R-, S-Xpemp [Xpemp = (4-X-1-phenylethylamino)methylphosphonic acid, X = H, F, Cl, Br] systems and study how the weak interactions influence the formation of layered or tubular structures. Four pairs of homochiral isostructural compounds R-, S-Co(Xpemp)(H2O)2 [X = H (1H), F (2F), Cl (3Cl), Br (4Br)] were obtained with tubular structures. The inclusion of 3,3'-azobipyridine (ABP) guest molecules led to compounds R-, S-[Co(Xpemp)(H2O)2]4·ABP·H2O with layered structures when X was Cl (5Cl) and Br (6Br), but tubular compounds 1H and 2F when X was H and F. Layered structures were also obtained for racemic compounds meso-Co(Xpemp)(H2O)2 [X = F (7F), Cl (8Cl), Br (9Br)] using racemic XpempH2 as the reaction precursor, but not when X = H. A detailed study on R-6Br revealed that layer-to-tube transformation occurred upon removal of ABP under hydrothermal conditions, forming R-4Br with a tubular structure. Similar layer-to-tube conversion did not occur in organic solvents. The results demonstrate that weak interlayer interactions are a prerequisite but not sufficient for the rolling-up of the layers. In the present cases, water also provides a driving force in the layer-to-tube transformation. The experimental results were rationalized by theoretical calculations.

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae.

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3'-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5' of the stop codon, six nucleotides 3' of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3'-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3'-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3'-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

The DHODH inhibitor PTC299 arrests SARS-CoV-2 replication and suppresses induction of inflammatory cytokines.

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-COV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally bioavailable compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS-COV-2 replication (EC50 range, 2.0-31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17 F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.

Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination.

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).

The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines.

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-CoV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally available compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS CoV-2 replication (EC 50 range, 2.0 to 31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.

Synthesis of the Elusive S = 1/2 Star Structure: A Possible Quantum Spin Liquid Candidate.

Materials with two-dimensional, geometrically frustrated, spin-1/2 lattices provide a fertile playground for the study of intriguing magnetic phenomena such as quantum spin liquid (QSL) behavior, but their preparation has been a challenge. In particular, the long-sought, exotic spin-1/2 star structure has not been experimentally realized to date. Here we report the synthesis of [(CH3)2(NH2)]3[CuII3(µ3-OH)(µ3-SO4)(µ3-SO4)3]·0.24H2O with an S = 1/2 star lattice. On the basis of the magnetic susceptibility and heat capacity measurements, the layered Cu-based compound exhibits antiferromagnetic interactions but no magnetic ordering or spin freezing down to 2 K. The spin-frustrated material appears to be a promising QSL candidate.

A Mixed-Valent Iron (II/III) Diamond Chain with Single-Ion Anisotropy.

The geometrically frustrated diamond spin chain system has yielded materials with a diversity of interesting magnetic properties but is predominantly limited to compounds with single-spin components. Here, we report the compound [(CH3)2NH2]6[FeIII4FeII2(µ3-O)2(µ3-OH)2(µ3-SO4)8] (1), which features the mixed-valent iron(II/III) diamond chain: ∞[FeIII-(FeIII)2-FeIII-(FeII)2]. 57Fe Mössbauer spectroscopy shows that two-thirds of the total spins in the ∞[FeIII4FeII2] diamond chain are spin-5/2 (high-spin FeIII), while the remaining one-third are spin-2 (high-spin FeII). To date, 1 is the only diamond-chain compound composed of more than one type of dimer, namely, (FeIII)2 and (FeII)2. On the basis of temperature-dependent 57Fe Mössbauer spectroscopy data, an alternating noncollinear 90° magnetic structure is proposed. Both the (FeIII)2 and (FeII)2 dimers are antiferromagnetically coupled and align in the direction along the chain axis ≈ [010], whereas the moments of the bridging FeIII monomers are oriented orthogonally. The spin canting, arising from the anisotropy of the FeII ions, leads to ferrimagnetic ordering at low temperatures.

Sulfate-Bridged, Octanuclear Chromic and Ferric Wheels.

Two new amine-templated transition metal-based sulfates, [(CH3)2NH2]17.4 [SO4]0.7 [MIII8(µ2-OH)8(µ2-SO4)16] where M = Cr and Fe, have been synthesized via mild solvothermal synthesis. The compounds are isostructural and were refined in the monoclinic space group P21/n. They feature the rare sulfate-bridged inorganic molecular wheels [CrIII8(OH)8(SO4)16]16- and [FeIII8(OH)8(SO4)16]16-. In both the octanuclear chromic (J = -2.4 cm-1 based on Hex = -J Sî · Sĵ convention) and ferric wheels (J = -38.3 cm-1), the coupling between the adjacent metal ions is antiferromagnetic giving spin-singlet ground states. The variation in the magnitude of the exchange coupling constants is due to the differences in the superexchange mechanisms, namely, a π-pathway for the Cr- and a σ-pathway for the Fe-wheel cluster.

Correction: Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

[This corrects the article DOI: 10.1371/journal.pgen.1007806.].

Targeting of Hematologic Malignancies with PTC299, A Novel Potent Inhibitor of Dihydroorotate Dehydrogenase with Favorable Pharmaceutical Properties.

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.

Selective profiling of ribosomes associated with yeast Upf proteins.

Ribosomes associated with nonsense-mediated decay factors Upf1, Upf2, or Upf3 were purified by immunoprecipitation, and enrichment and stoichiometry of Upf factors and ribosomal proteins were analyzed by western blot and mass spectrometry. Using a small RNA library preparation protocol that eliminates in-gel RNA and cDNA size selection and incorporates four random nucleotides on each side of the ribosome-protected RNA fragment allowed recovery, detection, and analysis of all size classes of protected fragments from a sample simultaneously.