Accurate gene regulation is essential for development and homeostasis and dysregulated gene regulation is associated with human disease. At the post-transcriptional level, a variety of ribonuclease enzymes are involved in messenger RNA quality control and messenger RNA degradation. This project will focus on the biochemical characterisation of human ribonuclease enzymes involved in the degradation of messenger RNA. For example, the project will focus on the multi-component Ccr4-Not and Pan2-Pan3 ribonucleases, which have a preference for the degradation of poly(A) stretches, which are found at the 3' end of messenger RNA. The project will focus on the development of novel assays to analyse the properties of the ribonucleases using purified proteins. In addition, cell-based reporter assays will be used as a complementary approach. The assays may be used to test the activity of drug-like inhibitors of the Ccr4-Not deadenylase.
Double stranded RNA does not occur naturally in cells therefore when double stranded RNA is recognized by cells it is degraded into shorter fragments of 21-22 nucleotides in length by a specific complex within the cell (RNA induced silencing complex), the degraded fragments then binds to the complementary base pairs of the target mRNA thereby reducing the activity of the mRNA. This mechanism was later shown to be conserved in mammals. Short interfering (si)RNAs can be artificially introduced into cells to utilise this pathway with substantial therapeutic potential. However significant obstacles prevent clinical use. Therefore delivery vehicles based on polymers are required. This project has been performed to determine if a series of ABA tri-block polymers synthesized from poly (ethylene glycol) methyl ethermethacrylate (PEGMA475) and N, N- dimethylaminoethyl methacrylates (DMAEMA) using Reversible Addition Fragmentation Chain Transfer (RAFT) polymerisation can protect and deliver siRNA for gene silencing. The polymers differed by inclusion of varying amount of cholesterol in the PEGMA475 blocks from no cholesterol to 20%. The polymers were examined in a broad range of assays including the effect on cell viability, the ability to protect siRNA from serum degradation, cellular siRNA uptake and the uptake mechanism, cell localisation and gene silencing. The results from this project show that the polymers were able to efficiently bind siRNA to produce polymer/siRNA nanoparticles varying considerably in size, which is believed to be due to the inclusion of cholesterol in the PGMA blocks. All polymers facilitated entry of the nanoparticles into cells, with the polymer containing 20% cholesterol being significantly more efficient. This polymer was also shown to enter the cells via macropinocytosis were it was co localized in lysosomes. Ultimately this polymer showed improved silencing in one cell line, however, further improvements are required for this polymer to be suitable for clinical use.