Pervasive translation of Xrn1-sensitive unstable long noncoding RNAs in yeast.

Despite being predicted to lack coding potential, cytoplasmic long noncoding (lnc)RNAs can associate with ribosomes. However, the landscape and biological relevance of lncRNA translation remain poorly studied. In yeast, cytoplasmic Xrn1-sensitive unstable transcripts (XUTs) are targeted by nonsense-mediated mRNA decay (NMD), suggesting a translation-dependent degradation process. Here, we report that XUTs are pervasively translated, which impacts their decay. We show that XUTs globally accumulate upon translation elongation inhibition, but not when initial ribosome loading is impaired. Ribo-seq confirmed ribosomes binding to XUTs and identified ribosome-associated 5'-proximal small ORFs. Mechanistically, the NMD-sensitivity of XUTs mainly depends on the 3'-untranslated region length. Finally, we show that the peptide resulting from the translation of an NMD-sensitive XUT reporter exists in NMD-competent cells. Our work highlights the role of translation in the posttranscriptional metabolism of XUTs. We propose that XUT-derived peptides could be exposed to natural selection, while NMD restricts XUT levels.

Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer.

Long noncoding (lnc)RNAs modulate gene expression alongside presenting unexpected source of neoantigens. Despite their immense interest, their ability to be transferred and control adjacent cells is unknown. Extracellular Vesicles (EVs) offer a protective environment for nucleic acids, with pro and antitumourigenic functions by controlling the immune response. In contrast to extracellular nonvesicular RNA, few studies have addressed the full RNA content within human fluids' EVs and have compared them with their tissue of origin. Here, we performed Total RNA-Sequencing on six Formalin-Fixed-Paraffin-Embedded (FFPE) prostate cancer (PCa) tumour tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. UEVs contain simplified transcriptome with intron-free cytoplasmic transcripts and enriched lnc/circular (circ)RNAs, strikingly common to an independent 20 patients' urinary cohort. Our full cellular and EVs transcriptome comparison within three PCa cell lines identified a set of overlapping 14 uEV-circRNAs characterized as essential for prostate cell proliferation in vitro and 28 uEV-lncRNAs belonging to the cancer-related lncRNA census (CLC2). In addition, we found 15 uEV-lncRNAs, predicted to encode 768 high-affinity neoantigens, and for which three of the encoded-ORF produced detectable unmodified peptides by mass spectrometry. Our dual analysis of EVs-lnc/circRNAs both in urines' and in vitro's EVs provides a fundamental resource for future uEV-lnc/circRNAs phenotypic characterization involved in PCa.

Direct Base-Assisted C‒H Cyclonickelation of 6-Phenyl-2,2'-bipyridine.

The organonickel complexes [Ni(Phbpy)X] (X = Br, OAc, CN) were obtained for the first time in a direct base-assisted arene C(sp2)-H cyclometalation reaction from the rather unreactive precursor materials NiX2 and HPhbpy (6-phenyl-2,2'-bipyridine) or from the versatile precursor [Ni(HPhbpy)Br2]2. Different from previously necessary C‒Br oxidative addition at Ni(0), an extended scan of reaction conditions allowed quantitative access to the title compound from Ni(II) on synthetically useful timescales through base-assisted C‒H activation in nonpolar media at elevated temperature. Optimisation of the reaction conditions (various bases, solvents, methods) identified 1:2 mixtures of acetate and carbonate as unrivalled synergetic base pairs in the optimum protocol that holds promise as a readily usable and easily tuneable access to a wide range of direct nickelation products. While for the base-assisted C‒H metalation of the noble metals Ru, Ir, Rh, or Pd, this acetate/carbonate method has been established for a few years, our study represents the leap into the world of the base metals of the 3d series.

Nickel-Alkyl Complexes with a Reactive PNC-Pincer Ligand.

Based on previous work related to the design and application of rigid tridentate phosphine-pyridine-phenyl coordination offered by a PNC-pincer ligand upon cyclometalation to nickel, the synthesis, spectroscopic and solid state characterization and redox-reactivity of two NiII(PNC) complexes featuring either a methyl (2CH3 ) or CF3 co-ligand (2CF3 ) are described. One-electron oxidation is proposed to furnish C-C reductive elimination, as deduced from a combined chemical, electrochemical, spectroscopic and computational study. One-electron reduction results in a ligand-centered radical anion, as supported by electrochemistry, UV spectroelectrochemistry, EPR spectroscopy, and DFT calculations. This further attenuates the breadth of chemical reactivity offered by such PNC-pincer ligands.

Passive On-Chip Superconducting Circulator Using a Ring of Tunnel Junctions.

We present the design of a passive, on-chip microwave circulator based on a ring of superconducting tunnel junctions. We investigate two distinct physical realizations, based on Josephson junctions (JJs) or quantum phase slip elements (QPS), with microwave ports coupled either capacitively (JJ) or inductively (QPS) to the ring structure. A constant bias applied to the center of the ring provides an effective symmetry breaking field, and no microwave or rf bias is required. We show that this design offers high isolation, robustness against fabrication imperfections and bias fluctuations, and a bandwidth in excess of 500 MHz for realistic device parameters.

Insulating Josephson Junction Chains as Pinned Luttinger Liquids.

Quantum physics in one spatial dimension is remarkably rich, yet even with strong interactions and disorder, surprisingly tractable. This is due to the fact that the low-energy physics of nearly all one-dimensional systems can be cast in terms of the Luttinger liquid, a key concept that parallels that of the Fermi liquid in higher dimensions. Although there have been many theoretical proposals to use linear chains and ladders of Josephson junctions to create novel quantum phases and devices, only modest progress has been made experimentally. One major roadblock has been understanding the role of disorder in such systems. We present experimental results that establish the insulating state of linear chains of submicron Josephson junctions as Luttinger liquids pinned by random offset charges, providing a one-dimensional implementation of the Bose glass, strongly validating the quantum many-body theory of one-dimensional disordered systems. The ubiquity of such an electronic glass in Josephson-junction chains has important implications for their proposed use as a fundamental current standard, which is based on synchronization of coherent tunneling of flux quanta (quantum phase slips).

Unsymmetrical N-Aryl-1-(pyridin-2-yl)methanimine Ligands in Organonickel(II) Complexes: More Than a Blend of 2,2'-Bipyridine and N,N-Diaryl-α-diimines?

The new organonickel complexes [(R-PyMA)Ni(Mes)X] [R-PyMA = N-aryl-1-(pyridin-2-yl)methanimine; aryl = phenyl, 2,6-Me2-, 3,5-Me2-, 2,4,6-Me3-, 2,6-iPr2-, 3,5-(OMe)2-, 2-NO2-4-Me-, 4-NO2-, 2-CF3-, and 2-CF3-6-F-phenyl; Mes = 2,4,6-trimethylphenyl; X = F, Cl, Br, or I] were obtained as approximate 1/1 cis and trans isomeric mixtures or pure cis isomers depending on the PyMA ligand and X. The [(R-PyMA)Ni(Mes)X] complexes with X = Br or Cl were directly synthesized from the precursors trans-[(PPh3)2Ni(Mes)X], while [(PyMA)Ni(Mes)X] derivatives with X = F or I were obtained from [(PyMA)Ni(Mes)Br] through X exchange reactions. Although density functional theory (DFT) calculations show a preference for the sterically favored cis isomers, both isomers could be observed in many cases; in three cases, even single crystals for X-ray diffraction could be obtained for the trans isomers. Possible intermediates for the isomerization were investigated by DFT calculations. All complexes were studied by multiple spectroscopic means, electrochemistry, and spectroelectrochemistry (for the reduction processes). The long-wavelength metal-to-ligand charge-transfer (MLCT) absorptions vary markedly with the R substituent of the ligand and the cathodic electrochemical potentials to a far smaller degree. Both are almost invariable upon variation of X. All of this is in line with Ni-based and π*-based lowest unoccupied molecular orbitals (LUMOs). In line with the unsymmetric character of the NPy^Nmethanimine ligand, electrochemistry and MLCT transitions seem to not correspond to the same type of π* LUMO, making these PyMA ligands more interesting than the symmetric heteroaromatic polypyridine ligands such as 2,2'-bipyridine (bpy; NPy^NPy) and N,N-diaryl-substituted aliphatic α-diimines (Nmethanimine^Nmethanimine) such as the diaza-1,3-butadienes (DAB). First attempts to use these complexes in Negishi-type cross-coupling reactions were successful.

Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure.

Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3' single-stranded (3'-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3'-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.

Charge filling factors in clean and disordered arrays of tunnel junctions.

We simulate one-dimensional arrays of tunnel junctions using the kinetic Monte Carlo method to study charge filling behaviour in the large charging energy limit. By applying a small fixed voltage bias and varying the offset voltage, we investigate this behaviour in clean and disordered arrays (both weak and strong disorder effects). The offset voltage dependent modulation of the current is highly sensitive to background charge disorder and exhibits substantial variation depending on the strength of the disorder. We show that while small fractional charge filling factors are likely to be washed out in experimental devices due to strong background charge disorder, larger factors may be observable.

Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma.

The mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomitant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra- and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation.

Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines.

The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.

Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

Relationships linking amplification level to gene over-expression in gliomas.

BACKGROUND: Gene amplification is thought to promote over-expression of genes favouring tumour development. Because amplified regions are usually megabase-long, amplification often concerns numerous syntenic or non-syntenic genes, among which only a subset is over-expressed. The rationale for these differences remains poorly understood. METHODOLOGY/PRINCIPAL FINDING: To address this question, we used quantitative RT-PCR to determine the expression level of a series of co-amplified genes in five xenografted and one fresh human gliomas. These gliomas were chosen because we have previously characterised in detail the genetic content of their amplicons. In all the cases, the amplified sequences lie on extra-chromosomal DNA molecules, as commonly observed in gliomas. We show here that genes transcribed in non-amplified gliomas are over-expressed when amplified, roughly in proportion to their copy number, while non-expressed genes remain inactive. When specific antibodies were available, we also compared protein expression in amplified and non-amplified tumours. We found that protein accumulation barely correlates with the level of mRNA expression in some of these tumours. CONCLUSIONS/SIGNIFICANCE: Here we show that the tissue-specific pattern of gene expression is maintained upon amplification in gliomas. Our study relies on a single type of tumour and a limited number of cases. However, it strongly suggests that, even when amplified, genes that are normally silent in a given cell type play no role in tumour progression. The loose relationships between mRNA level and protein accumulation and/or activity indicate that translational or post-translational events play a key role in fine-tuning the final outcome of amplification in gliomas.

Extrachromosomal amplification mechanisms in a glioma with amplified sequences from multiple chromosome loci.

Accumulation of extrachromosomal DNA molecules (double minute) is often responsible for gene amplification in cancers, but the mechanisms leading to their formation are still largely unknown. By using quantitative PCR, chromosome walking, in situ hybridization on metaphase chromosomes and whole genome analysis, we studied a glioma containing four extrachromosomally amplified loci (7p11, 1q32.1, 5p15 and 9p2). Complex extrachromosomal DNA molecules were formed by the fusion of several syntenic or non-syntenic DNA fragments from 7p11, 5p15 to 9p2. Fragments ranged from a few base pairs to megabase pairs. Scars of the amplification process remained at the original locus in the form of deletions or chromosome rearrangements. Chromosome fragmentation, due to replication stress, could explain this complex situation. In contrast, at 1q32.1, the initial extrachromosomal DNA molecule resulted from the circularization of a single fragment associated with an intrachromosomal deletion including, but larger than, the amplified sequence. The nature of the sequences involved in these rearrangements suggests that a V(D)J-like illegitimate recombination contributes to its formation.

Gefitinib and chemotherapy combination studies in five novel human non small cell lung cancer xenografts. Evidence linking EGFR signaling to gefitinib antitumor response.

The epidermal growth factor receptor (EGFR) signaling pathway is often activated in NSCLC, and thus represents a promising therapeutic target. We studied the antitumor activity of gefitinib (Iressa), an orally active EGFR-tyrosine kinase inhibitor, alone and in combination with standard chemotherapy in 5 recently established human NSCLC xenografts with wild-type EGFR. Mice were treated with 2 protocols of chemotherapy based on cisplatin (CDDP) combined with either gemcitabine (GEM) or vinorelbine (VNR). Gefitinib alone significantly inhibited tumor growth (TGI) in 4 of the 5 tumor xenografts (mean TGI of 58%, range: 25-70%). CDDP+VNR alone failed to achieve any significant responses, while CDDP+GEM achieved significant responses in 2 xenografts (TGI of 93 and 47%). Addition of gefitinib to CDDP+GEM potentialized chemotherapy in the 3 CDDP+GEM-resistant xenografts, but did not potentialize the CDDP+VNR combination. The effect of gefitinib treatment on the activity of extra cellular-regulated kinase (Erk), Akt, JNK and p38 kinases was assessed in IC9LC11 and IC1LC131, two NSCLC xenografts selected for their sensitivity and resistance to gefitinib, respectively. In IC9LC11, gefitinib strongly inhibited Erk, Akt and Jnk phosphorylation, but P38 remained active. Inversely, in IC1LC131, Erk and Akt pathways remained active, while Jnk and P38 pathways were inhibited by gefitinib. The data indicate that the antitumor activity of gefitinib in NSCLC, alone or in combination with chemotherapy, is tumor-dependent and is influenced by downstream signaling events independent of EGFR status.

Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability.

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.

Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.

Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.