RNA Whole-Mount In Situ Hybridization Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single-cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogeneous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos. © 2017 by John Wiley & Sons, Inc.

A newly identified Rab-GDI paralogue has a role in neural development in amphibia.

Vesicle shuttling is critical for many cellular and organismal processes, including embryonic development. GDI proteins contribute to vesicle shuttling by regulating the activity of Rab GTPases, controlling their cycling between the inactive cytosol and active membrane bound states. While identifying genes controlled by A-form DNA sequences we discovered a previously unknown member of the GDI family, GDI3. The GDI3 gene is found only in amphibians and fish and is developmentally expressed in Xenopus from neurula stages onwards in the neural plate, and subsequently in both dorsal and anterior structures. Depletion or over-expression of the GDI3 protein in Xenopus embryos gives rise to very similar phenotypes, suggesting that strict control of GDI3 protein levels is required for correct embryonic development. Our analysis suggests the evolutionary origins of GDI3 and that it is functionally distinct from GDI1. Predicted structural analysis of GDI3 suggests that the key difference between GDI1 and GDI3 lies in their lipid binding pockets.

RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation-Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.

Circular Dichroism for the Analysis of Protein-DNA Interactions.

The aim of this chapter is to provide information on the practical aspects of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) in protein-nucleic acids interaction solution studies. The chapter will describe the guidelines appropriate to designing experiments and conducting correct data interpretation, the use of both benchtop and synchrotron CD approaches is discussed and the advantages of SRCD outlined. Further information and a good general review of the field a can be found in Gray (Circular Dichroism of protein-nucleic acid interactions. In: Fasman GD (ed) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York. pp 469-500, 1996).

Omega 3 (n-3) fatty acids down-regulate nuclear factor-kappa B (NF-κB) gene and blood cell adhesion molecule expression in patients with homozygous sickle cell disease.

Chronic inflammation and reduced blood levels of omega-3 fatty acids (n-3) are known characteristics of sickle cell disease (SCD).The anti-inflammatory properties of n-3 fatty acids are well recognized. Omega-3 treated (n = 24), hydroxyurea (HU) treated (n = 18), and n-3 untreated (n=21) homozygous SCD patients (HbSS) and healthy (HbAA) controls (n = 25) matched for age (5-16 years), gender and socioeconomic status were studied. According to age (5-10) or (11-16) years, two or three capsules containing 277.8 mg docosahexaenoic (DHA) and 39.0mg eicosapentaenoic (EPA) or high oleic acid placebo (41%) were assigned to n-3 treated and n-3 untreated groups, respectively. Hydroxyurea treated group was on dosage more than 20 mg/kg/day. The effect of supplementation on systemic and blood cell markers of inflammation was investigated. The n-3 treated group had higher levels of DHA and EPA (p < 0.001) and lower white blood cell count and monocyte integrin (p < 0.05) compared with the n-3 untreated. No difference was detected between the two groups regarding C-reactive protein, granulocytes integrin and selectin, plasma tumour necrosis factor-α and interleukin-10. The n-3 treated group had lowered nuclear factor-kappa B (NF-κB) gene expression compared to n-3 untreated and HU treated groups (p < 0.05). This study provides evidence that supplementation with n-3 fatty acids may ameliorate inflammation and blood cell adhesion in patients with SCD.

An old dog and new tricks: Genetic analysis of a Tudor dog recovered from the Mary Rose wreck.

The Tudor warship the Mary Rose sank in the Solent waters between Portsmouth and the Isle of Wight on the 19th of July 1545, whilst engaging a French invasion fleet. The ship was rediscovered in 1971 and between 1979 and 1982 the entire contents of the ship were excavated resulting in the recovery of over 25,000 objects, including the skeleton of a small to medium sized dog referred to as the Mary Rose Dog (MRD). Here we report the extraction and analysis of both mitochondrial and genomic DNA from a tooth of this animal. Our results show that the MRD was a young male of a terrier type most closely related to modern Jack Russell Terriers with a light to dark brown coat colour. Interestingly, given the antiquity of the sample, the dog was heterozygotic for the SLC2A9 gene variant that leads to hyperuricosuria when found in modern homozygotic animals. These findings help shed light on a notable historical artefact from an important period in the development of modern dog breeds.

APTE: identification of indirect read-out A-DNA promoter elements in genomes.

BACKGROUND: Transcriptional regulation is normally based on the recognition by a transcription factor of a defined base sequence in a process of direct read-out. However, the nucleic acid secondary and tertiary structure can also act as a recognition site for the transcription factor in a process known as indirect read-out, although this is much less understood. We have previously identified such a transcriptional control mechanism in early Xenopus development where the interaction of the transcription factor ilf3 and the gata2 promoter requires the presence of both an unusual A-form DNA structure and a CCAAT sequence. Rapid identification of such promoters elsewhere in the Xenopus and other genomes would provide insight into a less studied area of gene regulation, although currently there are few tools to analyse genomes in such ways. RESULTS: In this paper we report the implementation of a novel bioinformatics approach that has identified 86 such putative promoters in the Xenopus genome. We have shown that five of these sites are A-form in solution, bind to transcription factors and fully validated one of these newly identified promoters as interacting with the ilf3 containing complex CBTF. This interaction regulates the transcription of a previously uncharacterised downstream gene that is active in early development. CONCLUSIONS: A Perl program (APTE) has located a number of potential A-form DNA promotor elements in the Xenopus genome, five of these putative targets have been experimentally validated as A-form and as targets for specific DNA binding proteins; one has also been shown to interact with the A-form binding transcription factor ilf3. APTE is available from http://www.port.ac.uk/research/cmd/software/ under the terms of the GNU General Public License.

A-form DNA structure is a determinant of transcript levels from the Xenopus gata2 promoter in embryos.

We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.

Methylation of Xilf3 by Xprmt1b alters its DNA, but not RNA, binding activity.

Modification of proteins by methylation has emerged as a key regulatory mechanism in many cellular processes, including gene control. Eighty to ninety percent of the arginine methylation in the cell is performed by the protein arginine methyl transferase PRMT1. ILF3, a protein involved in gene regulation at several levels, has been shown to be a substrate and regulator of PRMT1 in mammals. Here we show that the Xenopus orthologue of ILF3 (Xilf3) is methylated in vivo, and, at least in vitro, this methylation is carried out by Xprmt1b. The in vitro methylation of Xilf3 inhibits its ability to bind to DNA while leaving RNA binding activity unaltered. Consistent with these activities having a role in vivo, the DNA binding activity of the Xilf3-containing CBTF complex and the transcription of its target gene, Xgata2, are both decreased by overexpression of Xprmt1b in embryos. However, in contrast to other RNA binding proteins, a changing degree of methylation does not alter the subcellular localization of Xilf3. Several other proteins involved in gene regulation can bind both RNA and DNA; these data demonstrate a mechanism by which such binding activities may be controlled independently.

Identification of a structural and functional domain in xNAP1 involved in protein-protein interactions.

xNAP1 (Xenopus nucleosome assembly protein) belongs to the family of nucleosome assembly proteins (NAPs) and shares 92% identity with human and mouse NAP1. NAPs have been reported to have a role in nucleosome assembly, cell cycle regulation, cell proliferation and transcriptional control, although the precise function of NAP1 is still not clear. Here we report the identification of a putative domain of xNAP1 by limited proteolysis. This domain has been mapped in the xNAP1 protein sequence to residues 38-282 and thus lacks the acidic sequences at the N- and C-termini. We have studied this domain and related fragments in vitro and by a functional assay involving over-expression of the protein in Xenopus laevis embryos. Analytical ultracentrifugation shows that removal of the acidic N- and C-terminal regions does not prevent the formation of larger multimers, which are predominantly hexadecamers. Injection of mRNA encoding the full-length xNAP1 or the putative domain and other related constructs into Xenopus embryos gave identical phenotypes. These results are discussed in relation to protein-protein interactions between NAP1 octamers and a possible 'squelching' mechanism.

Intact RNA-binding domains are necessary for structure-specific DNA binding and transcription control by CBTF122 during Xenopus development.

CBTF122 is a subunit of the Xenopus CCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122 containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (an in vivo target of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA binding in vitro. In addition, these mutations alter the ability of CBTF122 fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 gene in vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding both in vitro and in vivo.

Structures of CUG repeats in RNA. Potential implications for human genetic diseases.

Triplet repeats that cause human genetic diseases have been shown to exhibit unusual compact structures in DNA, and in this paper we show that similar structures exist in shorter "normal length" CNG RNA. CUG and control RNAs were made chemically and by in vitro transcription. We find that "normal" short CUG RNAs migrate anomalously fast on non-denaturing gels, compared with control oligos of similar base composition. By contrast, longer tracts approaching clinically relevant lengths appear to form higher order structures. The CD spectrum of shorter tracts is similar to triplex and pseudoknot nucleic acid structures and different from classical hairpin spectra. A model is outlined that enables the base stacking features of poly(r(G-C))(2).poly(r(U)) or poly(d(G-C))(2).poly(d(T)) triplexes to be achieved, even by a single 15-mer.