The role of Y-box binding protein 1 in prostate cancer

This thesis examined the possible role of Y-box binding protein 1 (YBX1) in prostate cancer aggression and spread. Novel roles were uncovered for YBX1 in the regulation of several genes previously implicated in prostate cancer, as well as showing an effect for YBX1 in increasing tumour cell invasion and movement and reciprocal regulation of androgen-regulated gene networks. In addition, it was found that Y-box 1 regulated several other well-known cancer genes implicated in breast and other cancers. The work performed in this thesis has strengthened the foundations for pursuing YBX1 as a possible central target molecule in prostate cancer therapeutics.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of doublestranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 59 thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.

Carrot mottle mimic virus (CMoMV): A second umbravirus associated with carrot motley dwarf disease recognised by nucleic acid hybridisation

Carrot mottle umbravirus (CMoV) has always been found co-infecting plants with carrot red leaf luteovirus (CRLV) and in carrot (Daucus carota) these co-infections are associated with carrot motley dwarf disease (CMD). CMD occurs wherever carrots are grown. Hence, CMoV was believed to have a corresponding global distribution. However, little or no hybridisation was detected between cDNA generated from the sequenced Australian isolate of CMoV (CMoV-A) and RNA from the much studied Scottish isolate of CMoV (CMoV-S). A weak hybridisation signal was obtained using cDNA to a conserved part of the RNA-dependent RNA polymerase gene of CMoV-A, but when cDNAs to other parts of the CMoV-A genome were used as probes there was no detectable hybridisation with CMoV-S RNA. This lack of hybridisation suggests that the two virus isolates have relatively divergent genomes and that they should be regarded as distinct virus species. Both viruses are transmitted by Cavariella aegopodii, but only with the help of CRLV, and they yield almost identical double-stranded RNA profiles. For these reasons, we propose that the CMoV isolate from Australia be renamed carrot mottle mimic umbravirus (CMoMV). cDNA to CMoMV RNA hybridised with RNA from an isolate from New Zealand, whereas cDNA to CMoV-S RNA hybridised with RNA from isolates from England and Morocco but not to RNA from the isolate from New Zealand. Although preliminary, these data suggest that CMoV and CMoMV may have different global distributions.

Complete genomic sequence of a Rubus yellow net virus isolate and detection of genome-wide pararetrovirus-derived small RNAs

Rubus yellow net virus (RYNV) was cloned and sequenced from a red raspberry (Rubus idaeus L.) plant exhibiting symptoms of mosaic and mottling in the leaves. Its genomic sequence indicates that it is a distinct member of the genus Badnavirus, with 7932. bp and seven ORFs, the first three corresponding in size and location to the ORFs found in the type member Commelina yellow mottle virus. Bioinformatic analysis of the genomic sequence detected several features including nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Subsequent sequencing of the small RNAs (sRNAs) from RYNV-infected R. idaeus leaf tissue was used to determine any RYNV sequences targeted by RNA silencing and identified abundant virus-derived small RNAs (vsRNAs). The majority of the vsRNAs were 22-nt in length. We observed a highly uneven genome-wide distribution of vsRNAs with strong clustering to small defined regions distributed over both strands of the RYNV genome. Together, our data show that sequences of the aphid-transmitted pararetrovirus RYNV are targeted in red raspberry by the interfering RNA pathway, a predominant antiviral defense mechanism in plants. © 2013.

Small RNA sequencing of potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein

Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3' proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5' transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ~500. nt from the 3' terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5' transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5' of PLRV genomic RNA. © 2013.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

Nucleic acid hybridizations as radioactive tool for rapid detection of banana bunchy top virus

Banana bunchy top disease (BBTD) caused by banana bunchy top virus (BBTV) was radioactively detected by nucleic acid hybridization techniques. Results showed that, 32P-labelled insert of pBT338 was hybridized with nucleic acid extracts from BBTV-infected plants from Egypt and Australia but not with those from CMV-infected plants from Egypt. Results revealed that BBTV was greatly detected in midrib, roots, meristem, corm, leaves and pseudostem respectively. BBTV was also detected in symptomless young plants prepared from diseased plant materials grown under tissue culture conditions but was not present in those performed from healthy plant materials. The sensitivity of dot blot and Southern blot hybridizations for the detection of BBTV was also performed for the detection of BBTV.

The Saccharomyces cerevisiae RNA-binding protein Rbp29 functions in cytoplasmic mRNA metabolism

Here we report that the Saccharomyces cerevisiae RBP29 (SGN1, YIR001C) gene encodes a 29-kDa cytoplasmic protein that binds to mRNA in vivo. Rbp29p can be co-immunoprecipitated with the poly(A) tail-binding protein Pab1p from crude yeast extracts in a dosageand RNA-dependent manner. In addition, recombinant Rbp29p binds preferentially to poly(A) with nanomolar binding affinity in vitro. Although RBP29 is not essential for cell viability, its deletion exacerbates the slow growth phenotype of yeast strains harboring mutations in the eIF4G genes TIF4631 and TIF4632. Furthermore, overexpression of RBP29 suppresses the temperaturesensitive growth phenotype of specific tif4631, tif4632, and pab1 alleles. These data suggest that Rbp29p is an mRNA-binding protein that plays a role in modulating the expression of cytoplasmic mRNA.

The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16–18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

Vaccination with an Insulin-like Growth Factor Binding Protein-1 (IGFBP-1)-bBased Vaccine Improves Growth in Feed-Restricted Brahman Steers

Dry-season weight loss in grazing cattle in northern Australia has been attenuated using a number of strategies (Hunter and Vercoe, 1987, Sillence et al. 1993, Gazzola and Hunter, 1999). Furthermore, the potential to improve efficiency of feed utilisation (and thus, dry-season performance) in ruminants through conventional modulation of the insulin-like growth factor (IGF) axis (Oddy and Owens, 1997, Hill et al., 1999) and through immunomodulation of the IGF axis (Hill et al., 1998a,b) has been demonstrated. The present study investigated the use of a vaccine directed against IGFBP-1 in Brahman steers which underwent a period of nutritional restriction followed by a return to wet-season grazing.

Differentiation of Bordetella avium and Related Species by Cellular Fatty Acid Analysis

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.

Small local variations in B-form DNA lead to a large variety of global geometries which can accommodate most DNA-binding protein motifs

An important question of biological relevance is the polymorphism of the double-helical DNA structure in its free form, and the changes that it undergoes upon protein-binding. We have analysed a database of free DNA crystal structures to assess the inherent variability of the free DNA structure and have compared it with a database of protein-bound DNA crystal structures to ascertain the protein-induced variations.

Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation of E. coli Maltose Binding Protein

SecB, a soluble cytosolic chaperone component of the Secexport pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (ΔTm =7( 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability ofMBP(ΔTm=1(0.4 K) or preMBP (ΔTm=0(0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils.Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can reatly influence protein stability and aggregation propensity.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

An uncommon nucleotide conformation shown by molecular structure of deoxyuridine-5-phosphate and nucleic acid stereochemistry

CRYSTAL structure determinations of nucleic acid fragments have shown that several of the conformational features found in the monomeric building blocks are also manifested at the nucleic acid level. Stereochemical variations between thymine and uracil nucleotides are therefore of interest as they can provide a structural basis for some of the differences between the conformations of DNA and RNA. X-ray studies have so far not shown any major dissimilarities between these two nucleotide species although the sugar ring of deoxyribonucleotides is found to possess greater flexibility than that in ribonucleotides. We report here the molecular structure of deoxyuridine-5'-phosphate (dUMP-5') which is not a common monomer unit of DNAs as it is replaced by its 5-methyl analogue deoxythymidine-5'-phosphate (dTMP-5'). The investigation was undertaken to help determine whether or not this implied a fundamental difference between the geometries of these two molecules.