Biochemical and structural characterization of the flavodoxin-like domain of the Schizosaccharomyces japonicus putative tRNA Phe 4-demethylwyosine synthase Tyw1 in complex with FMN.

The S-adenosyl-L-methionine-dependent tRNA 4-demethylwyosine synthase TYW1 catalyzes biosynthesis of 4-demethylwyosine (imG-14), the precursor for wyosine, the hypermodified guanine-derived nucleotide present at position 37 of phenylalanine tRNAs of archaea and eukarya. Eukaryotic TYW1 enzymes contain N-terminal flavodoxin-like and C-terminal radical-SAM domains. We determined co-crystal structures of the flavodoxin-like domain of the putative Tyw1 from Schizosaccharomyces japonicus in complex with flavin mononucleotide (FMN), exploiting an unexpected anomalous scatterer present in the recombinant protein. Our results show how eukaryotic TYW1 enzymes bind the coenzyme FMN and will help further elucidation of the structural enzymology of 4-demethylwyosine synthesis.

Binding between G Quadruplexes at the Homodimer Interface of the Corn RNA Aptamer Strongly Activates Thioflavin T Fluorescence.

Thioflavin T (ThT) is widely used for the detection of amyloids. Many unrelated DNAs and RNAs that contain G-quadruplex motifs also bind ThT and strongly activate its fluorescence. To elucidate the structural basis of ThT binding to G quadruplexes and its fluorescence turn-on, we determined its co-crystal structure with the homodimeric RNA Corn, which contains two G quadruplexes. We found that two ThT molecules bind in the dimer interface, constrained by a G quartet from each protomer into a maximally fluorescent planar conformation. The unliganded Corn homodimer crystal structure reveals a collapsed fluorophore-binding site. In solution, Corn must fluctuate between this and an open, binding-competent conformation. A co-crystal structure with another benzothiazole derivate, thiazole orange (TO), also shows binding at the Corn homodimer interface. As the bound ThT and TO make no interactions with the RNA backbone, their Corn co-crystal structures likely explain their fluorescence activation upon sequence-independent DNA and RNA G-quadruplex binding.

A homodimer interface without base pairs in an RNA mimic of red fluorescent protein.

Corn, a 28-nucleotide RNA, increases yellow fluorescence of its cognate ligand 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) by >400-fold. Corn was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, in which six unpaired adenosines break overall two-fold symmetry, lacks any intermolecular base pairs. The homodimer encapsulates one DFHO at its interprotomer interface, sandwiching it with a G-quadruplex from each protomer. Corn and the green-fluorescent Spinach RNA are structurally unrelated. Their convergent use of G-quadruplexes underscores the usefulness of this motif for RNA-induced small-molecule fluorescence. The asymmetric dimer interface of Corn could provide a basis for the development of mutants that only fluoresce as heterodimers. Such variants would be analogous to Split GFP, and may be useful for analyzing RNA co-expression or association, or for designing self-assembling RNA nanostructures.

Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4).

Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.

On the aggregation properties of FMRP--a link with the FXTAS syndrome?

Fragile X mental retardation protein (FMRP) is an RNA binding protein necessary for correct spatiotemporal control of neuronal gene expression in humans. Lack of functional FMRP causes fragile X mental retardation, which is the most common inherited neurodevelopmental disorder in humans. In a previous study, we described the biochemical and biophysical aggregation properties of constructs spanning the conserved region of FMRP and of two other human fragile X related (FXR) proteins, FXR1P and FXR2P. Here, we show that the same regions have an intrinsic tendency to aggregate and spontaneously misfold towards ß-rich structures, also under non-destabilizing conditions. These findings pave the way to future studies of the mechanism of formation of FXR-containing ribonucleoprotein granules and suggest a possible link with the as yet poorly understood FXR proteins' associated pathologies.

A study of the ultrastructure of fragile-X-related proteins.

Fragile-X-related proteins form a family implicated in RNA metabolism. Their sequence is composed of conserved N-terminal and central regions which contain Tudor and KH domains and of a divergent C-terminus with motifs rich in arginine and glycine residues. The most widely studied member of the family is probably FMRP (fragile X mental retardation protein), since absence or mutation of this protein in humans causes fragile X syndrome, the most common cause of inherited mental retardation. Understanding the structural properties of FMRP is essential for correlating it with its functions. The structures of isolated domains of FMRP have been reported, but nothing is yet known with regard to the spatial arrangement of the different modules, partly because of difficulties in producing both the full-length protein and its multidomain fragments in quantities, purities and monodispersity amenable for structural studies. In the present study, we describe how we have produced overlapping recombinant fragments of human FMRP and its paralogues which encompass the evolutionary conserved region. We have studied their behaviour in solution by complementary biochemical and biophysical techniques, identified the regions which promote self-association and determined their overall three-dimensional shape. The present study paves the way to further studies and rationalizes the existing knowledge on the self-association properties of these proteins.

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer.

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of gamma-filamin repeat 23.

Human gamma-filamin is a protein of 2705 amino-acid residues that localizes mainly in the myofibrillar Z-disc and to smaller extent in the subsarcolemmal region of striated muscle cells. gamma-Filamin consists of an N-terminal actin-binding domain followed by a long rod-shaped region. The rod-shaped region consists of 24 immunoglobulin-like domains that form a platform for interaction with different transmembrane, cell-signalling and cytoskeletal proteins. gamma-Filamin repeat 23 was indicated as being necessary for binding to the muscle-specific subsarcolemmal proteins gamma- and delta-sarcoglycan and the myofibrillar protein FATZ1. The recombinant gamma-filamin repeat 23 was crystallized using the hanging-drop vapour-diffusion method, which yielded needle-shaped diffraction-quality crystals. Diffraction data were collected to 2.05 angstroms resolution using 1.2 angstroms wavelength synchrotron radiation. Preliminary structural analysis shows one molecule, with predominantly beta secondary-structure elements, per asymmetric unit.