The 5′-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5′-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3′-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5′-strand of mivaRNAI. Of the small number of 5′-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3′-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5′-strand

Studies on nuclear RNA polymerases and the role of cyclic nucleotides on transcription

1. An assay for measuring the endogenous RNA polymerase activity in Tetrahymena pyriformis nuclei was developed. The reaction rate was linear for 60 minutes at 25°C. This rate of the reaction was dependent on the presence of four nucleoside triphosphates (ATP, GTP, CTP and UTP), Mn⁺⁺ or Mg⁺⁺ ions, and was optimal at high concentrations of KC1 or (NHO₄)₂80₄, (0.15 M). 2. α-amanitin (a specific inhibitor of RNA polymerase II) was used to differentiate between the different classes of nuclear RNA polymer­ases, RNA polymerase I and RNA polymerase II. 3.6 x 10⁻4 M α-amanitin inhibited approximately 601 of total RNA polymerase activity in nuclei isolated from logarithmically growing cells. Thus about two-thirds of the nuclear RNA polyiserasa activity was due to RNA polymerase II. 3. Cyclic AMP or dibutyryl cyclic AMP stimulated nuclear RNA polymerase activity at physiological concentrations (10⁻⁶ - 10⁻⁸ M). Maximum stimulation was obtained using 10⁻⁷ M cyclic AMP or 10⁻⁷ M dibutyryl cyclic AMP. The increase .in activity stimulated by cyclic AMP in isolated nuclei was dependent on salt concentration. Cyclic AMP stimulated endogenous RNA polymerase I and inhibited endogenous RNA polymerase II activity. Chromatin-bound RNA polymarase activity was also stimulated by this cyclic nucleotide. 4. Cyclic CMP or dibutyryl cyclic GMP in the presence of CaClg stimulated nuclear RNA polymerase activity at physiological concentrations (10⁻⁸8 - 10⁻10 M). Maximum stimulation of nuclear RNA polymerase activity by cyclic GMP in the presence of 2 mM CaCl2 occurred at 10“10 M cyclic nucleotide. 10”8 M dibutyryl cyclic GMP plus 2 mM CaCl2 produced maximum stimulation. The stimulation was CaCl2 dependent. 5 No significant stimulation of nuclear RNA polymerase activity was observed with 5'-AMP, 5'-GMP or 3',5'-cyclic CMP. 6. Chromatin-bound cyclic GMP and cyclic AMP phosphodiesterases (degrading enzymes of cyclic GMP and cyclic AMP respectively) were demonstrated to occur in Tetrahymana pyriformia nuclei. 7. The levels of total SNA polymerase activity were determined during the course of the natural cell cycle. By using α-amanitin, approximately two-thirds of the activity during the S phase of the cell cycle was found to be due to RNA polymerase II. RMA polymerase I activity was predominant in the G2 phase of the cell cycle. 8. An assay for endogenous protein kinase activity in Tetrahymena pyriformia nuclei was developed and validated. The cyclic AMP- dependent phosphorylation of nuclear proteins was found to be a salt- dependent process. Dibutyryl cyclic AMP stimulated the phosphorylation of the endogenous protein (s) and.exogenous substrates in a.partially purified nuclear fraction -containing RNA polymerase activity by 5-fold

The expression of biologically active recombinant ricin A chain "in vitro"

The major aim of this project was to attempt to define residues In rlcln A chain which are involved In the catalytic activity of the protein and to define a rlcin A chain molecule of minimum size which still remains active. A simple and sensitive system was developed in which the expression and assessment of biological activity of recombinant rlcln A chain are combined. This represents one of few reported examples of the ability to assess the activity of protein expressed from in vitro synthesised RHA in a cell free system. When recombinant ricln A chain transcripts were translated in a rabbit reticulocyte lysate, the ribosomes were rapidly inactivated. In contrast, ribosomes which have translated transcripts encoding non toxic polypeptides such as ricin B chain are not inactivated. Ribosome inactivation was accompanied by a highly specific modification of 28S rRNA which Is thought to cause the inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under conditions which inhibit reticulocyte ribosomes, confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inactivation by ricln A chain than are mammalian ribosomes. Using the same system, it was shown that by deleting an internal hexapeptide which shares homology with hamster EF 2, catalytic activity was completely abolished. Deleting a second Internal pentapeptide, conserved between ricin A chain and trichosanthln, had no effect. Deleting the first nine residues from the V terminus of ricln A chain did not affect toxicity, whereas deleting a further three residues Inactivated the polypeptide. Point mutations which Individually converted arginine 48 and arginine 56 to alanine residues or which removed arginine 56 were also without effect on the catalytic activity of the toxin

Investigating the Effect of Cell Culture Compositions on Mitochondrial Metabolism, Dynamics, and Transcriptome and Proteome of cells

The phytoestrogen Resveratrol (RES) is a natural polyphenol that has been detected in more than 70 plant species. RES has structural similarity to mammalian estrogens and can bind to estrogen receptors, eliciting genomic and non-genomic effects. Both RES and physiological estrogens like 17-β-estradiol (E2) have wide-ranging effects on mitochondria. In this thesis, I began by investigating RES’s effects on mitochondrial network dynamics (Chapter 2) and discovered a pro-fusion activity apparently mediated by the mitofusin enzyme Mfn2. RES stimulated mitochondrial network hyper-fusion morphology in all three cell lines investigated (C2C12 (mouse myoblast), PC3 (prostate cancer), and MEFs (mouse embryonic fibroblast)), but the effect was absent in Mfn2-null MEFs. As this work was being completed; several research groups introduced ‘physiologic cell culture media’ that are modeled on the human plasma metabolome. I co-authored a study (not in this thesis) demonstrating that RES’s effects on mitochondrial dynamics are dependent on cell culture conditions. To follow up on this, I investigated whether E2’s mitochondrial effects might also be dependent on the cell culture environment, and showed conclusively that this is indeed the case, using C2C12 cells as a model system (Chapter 3). These results and those published by others in 2017-2019 suggested that medium composition can profoundly affect cellular functions. In Chapter 4, I followed this up by studying how culture conditions affect mitochondrial bioenergetics and network morphology using four cancer cell lines and showed that this is a significant issue. Finally, to gain a more complete understanding of this phenomenon, I completed a full transcriptomic and proteomic analysis of media effects using MCF7 breast cancer cells as a model (Chapter 5). I showed that hundreds of transcripts and proteins are affected according to culture conditions. Taken together, the results presented in this thesis emphasize the significant extent to which the cell culture environment affects experimental outcomes, particularly with respect to mitochondrial bioenergetics and dynamics. This information contributes to the development of cell culture experiments providing results that can be translated in vivo

Investigation of protein-protein interactions: multibody docking, association/dissociation kinetics and macromolecular crowding

Protein-protein interactions are central to understanding how cells carry out their wide array of functions and metabolic procedures. Conventional studies on specific protein interactions focus either on details of one-to-one binding interfaces, or on large networks that require a priori knowledge of binding strengths. Moreover, specific protein interactions, occurring within a crowded macromolecular environment, which is precisely the case for interactions in a real cell, are often under-investigated. A macromolecular simulation package, called BioSimz, has been developed to perform Langevin dynamics simulations on multiple protein-protein interactions at atomic resolution, aimed at bridging the gaps between structural, kinetic and crowding studies on protein-protein interactions. Simulations on twenty-seven experimentally determined protein-protein interactions, indicated that the use of contact frequency information of proteins forming specific encounters can guide docking algorithms towards the most likely binding regions. Further evidence from eleven benchmarked protein interactions showed that the association rate constant of a complex, kon, can be estimated, with good agreement to experimental values, based on the retention time of its specific encounter. Performing these simulations with ten types of environmental protein crowders, it suggests, from the change of kon, that macromolecular crowding improves the association kinetics of slower-binding proteins, while it damps the association kinetics of fast, electrostatics-driven protein-protein interactions. It is hypothesised, based on evidence from docking, kinetics and crowding, that the dynamics of specific protein-protein encounters is vitally important in determining their association affinity. There are multiple factors by which encounter dynamics, and subsequently the kon, can be influenced, such as anchor residues, long-range forces, and environmental steering via crowders’ electrostatics and/or volume exclusion. The capacity of emulating these conditions on a common platform not only provides a holistic view of interacting dynamics, but also offers the possibility of evaluating and engineering protein-protein interactions from aspects that have never been opened before

Two initiation sites for adenovirus 5.5S RNA.

Adenovirus specific 5.5S RNA is heterogeneous at its 5' terminus. Complete pancreatic RNase digests of the RNA reveal a 5' terminal oligonucleotide (pp)pApGpCp in addition to the major 5' terminal (pp)pGpGpGpCp (1). Both 5' termini are detected early as well as late after adenovirus infection. In isolated nuclei, alpha-amanitin inhibits all 5.5S RNA transcription at a concentration of 200 microgram/ml, indicating that both initiation sites are recognized by RNA polymerase III