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.

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.

NMD: a multifaceted response to premature translational termination.

Although most mRNA molecules derived from protein-coding genes are destined to be translated into functional polypeptides, some are eliminated by cellular quality control pathways that collectively perform the task of mRNA surveillance. In the nonsense-mediated decay (NMD) pathway premature translation termination promotes the recruitment of a set of factors that destabilize a targeted mRNA. The same factors also seem to have key roles in repressing the translation of the mRNA, dissociating its terminating ribosome and messenger ribonucleoproteins (mRNPs), promoting the degradation of its truncated polypeptide product and possibly even feeding back to the site of transcription to interfere with splicing of the primary transcript.

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.

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.

Nonsense-Mediated mRNA Decay: Degradation of Defective Transcripts Is Only Part of the Story.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that monitors cytoplasmic mRNA translation and targets mRNAs undergoing premature translation termination for rapid degradation. From yeasts to humans, activation of NMD requires the function of the three conserved Upf factors: Upf1, Upf2, and Upf3. Here, we summarize the progress in our understanding of the molecular mechanisms of NMD in several model systems and discuss recent experiments that address the roles of Upf1, the principal regulator of NMD, in the initial targeting and final degradation of NMD-susceptible mRNAs. We propose a unified model for NMD in which the Upf factors provide several functions during premature termination, including the stimulation of release factor activity and the dissociation and recycling of ribosomal subunits. In this model, the ultimate degradation of the mRNA is the last step in a complex premature termination process.

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.

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.].

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.

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

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

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.

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.

High-resolution profiling of NMD targets in yeast reveals translational fidelity as a basis for substrate selection.

Nonsense-mediated mRNA decay (NMD) plays an important role in eukaryotic gene expression, yet the scope and the defining features of NMD-targeted transcripts remain elusive. To address these issues, we reevaluated the genome-wide expression of annotated transcripts in yeast cells harboring deletions of the UPF1, UPF2, or UPF3 genes. Our new RNA-seq analyses confirm previous results of microarray studies, but also uncover hundreds of new NMD-regulated transcripts that had escaped previous detection, including many intron-containing pre-mRNAs and several noncoding RNAs. The vast majority of NMD-regulated transcripts are normal-looking protein-coding mRNAs. Our bioinformatics analyses reveal that this set of NMD-regulated transcripts generally have lower translational efficiency and higher ratios of out-of-frame translation. NMD-regulated transcripts also have lower average codon optimality scores and higher transition probability to nonoptimal codons. Collectively, our results generate a comprehensive catalog of yeast NMD substrates and yield new insights into the mechanisms by which these transcripts are targeted by NMD.

Correction: Degradation of YRA1 Pre-mRNA in the Cytoplasm Requires Translational Repression, Multiple Modular Intronic Elements, Edc3p, and Mex67p.

[This corrects the article DOI: 10.1371/journal.pbio.1000360.].

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.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression.

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

Dissecting Porosity in Molecular Crystals: Influence of Geometry, Hydrogen Bonding, and [π···π] Stacking on the Solid-State Packing of Fluorinated Aromatics.

Porous molecular crystals are an emerging class of porous materials that is unique in being built from discrete molecules rather than being polymeric in nature. In this study, we examined the effects of molecular structure of the precursors on the formation of porous solid-state structures with a series of 16 rigid aromatics. The majority of these precursors possess pyrazole groups capable of hydrogen bonding, as well as electron-rich aromatics and electron-poor tetrafluorobenzene rings. These precursors were prepared using a combination of Pd- and Cu-catalyzed cross-couplings, careful manipulations of protecting groups on the nitrogen atoms, and solvothermal syntheses. Our study varied the geometry and dimensions of precursors, as well as the presence of groups capable of hydrogen bonding and [π···π] stacking. Thirteen derivatives were crystallographically characterized, and four of them were found to be porous with surface areas between 283 and 1821 m2 g-1. Common to these four porous structures were (a) rigid trigonal geometry, (b) [π···π] stacking of electron-poor tetrafluorobenzenes with electron-rich pyrazoles or tetrazoles, and