NF90 interacts with components of RISC and modulates association of Ago2 with mRNA.

BACKGROUND: Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. RESULTS: Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3' UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. CONCLUSIONS: These findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.

An integrated DNA and RNA variant detector identifies a highly conserved three base exon in the MAP4K5 kinase locus.

RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5V569E and MAP4K5V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.

The effects of p53 gene inactivation on mutant proteome expression in a human melanoma cell model.

BACKGROUND: The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape. METHODS: An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database. RESULTS: Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes. CONCLUSION: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells. GENERAL SIGNIFICANCE: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

NF90 modulates processing of a subset of human pri-miRNAs.

MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

Copper complexes of synthetic peptides mimicking neurotrophin-3 enhance neurite outgrowth and CREB phosphorylation.

In this work we report on the synthesis and physiochemical/biological characterization of a peptide encompassing the first thirteen residues of neurotrophin-3 (NT-3). The protein capability to promote neurite outgrowth and axonal branching by a downstream mechanism that involves the increase of the cAMP response element-binding level (CREB) was found for the NT3(1-13) peptide, thus validating its protein mimetic behaviour. Since copper ions are also involved in neurotransmission and their internalization may be an essential step in neuron differentiation and CREB phosphorylation, the peptide and its copper complexes were characterized by potentiometric and spectroscopic techniques, including UV-visible, CD and EPR. To have a detailed picture of the coordination features of the copper complexes with NT3(1-13), we also scrutinized the two peptide fragments encompassing the shorter sequences 1-5 and 5-13, respectively, showing that the amino group is the main anchoring site for Cu(ii) at physiological pH. The peptide activity increased in the presence of copper ions. The effect of copper(ii) addition is more marked for NT3(1-13) than the other two peptide fragments, in agreement with its higher affinity for metal ions. Confocal microscopy measurements carried out on fluorescently labelled NT3(1-13) indicated that copper ions increase peptide internalization.

Soluble sugar-based quinoline derivatives as new antioxidant modulators of metal-induced amyloid aggregation.

Oxidative stress and protein aggregation have been demonstrated to be the major factors involved in neurodegenerative diseases. Metal ions play a pivotal role, acting as mediators of neurotoxicity either by favoring or redox cycling. Thus, they represent a promising and suitable therapeutic target for the treatment of neurodegenerative disorders. In particular, the development of bifunctional or multifunctional molecules, which have antiaggregant and metal-chelating/antioxidant properties, may be considered as a valuable strategy for the treatment of neurodegeneration considering its multifactorial nature. Herein, we report the design and the characterization of four new multifunctional sugar-appended 8-hydroxyquinolines focusing on the effects of the conjugation with trehalose, a nonreducing disaccharide involved in the protection of proteins and cells against environmental stresses. These glycoconjugates do not exhibit any antiproliferative activity against three human cell lines of different histological origin, unlike 8-hydroxyquinolines. The multiple properties of the new derivatives are highlighted, reporting their Cu(2+) and Zn(2+) binding ability, and antioxidant and antiaggregant capacities. In particular, these latter were determined by different assays, including the evaluation of their ability to modulate or even suppress the aggregation of Aß1-40 and Aß1-42 peptides induced by copper or zinc ions.

Intramolecular weak interactions in the thermodynamic stereoselectivity of copper(II) complexes with carnosine-trehalose conjugates.

The interactions of metal ions with chiral molecules are of particular interest for relevant biochemical processes, as many of them are made possible only with a selected chirality of the stereocenters. In this work we report a study of the stereoselectivity of copper(II) complexes with D-trehalose-L-carnosine and D-trehalose-D-carnosine as a prototypical case of natural chirality selection. The interest in L-carnosine dipeptide is compounded by its antioxidant and antitumor properties, which are further enhanced when combined with D-trehalose. Potentiometric, calorimetric, and UV/circular dichroism (CD) spectroscopic measurements show that the copper(II) dimer of D-trehalose-L-carnosine is more stable than the D-trehalose-D-carnosine diastereoisomeric copper(II) dimer (log ß(L)(22-2) - log ß(D)(22-2) = 3.6). Free-energy calculations highlight that the cause of this different behavior lies with different intramolecular weak interactions between the diastereoisomers. The different pattern of hydrogen bonds and the different CH-π interactions between the π-electron-rich imidazole and the α-glucose rings are more favorable by 5 kcal mol(-1) in the L dimer.