RNA G-quadruplexes and polyamines effects on translation: data analysis and development of software for ribosome pause prediction
University College Cork
Ribosome profiling (Ribo-seq) technique provides a global snapshot of actively translating ribosomes by capturing the ribosome protected mRNA fragments. Ribo-seq technique can be implemented to study various translation aspects such as differential gene expression at the translation level, ribosomal frameshifting, decoding rate, ribosome pausing etc. The process of translation can be affected locally by the sequence of translated mRNA, e.g. by specific secondary structures as well as globally by cellular conditions, e.g. concentrations of metabolites such as polyamines. G-quadruplexes is a type of RNA secondary structures that has been implicated in mRNA translation. These G-quadruplex structures have been suggested to play roles in capdependent, cap-independent and repeat associated non-AUG (RAN) translation. Gquadruplex structures are also known to stimulate ribosomal frameshifting and ribosome pausing. In my work, I explored the effect of G-quadruplex on translation by utilizing the publicly available ribosome-profiling datasets to find the relationship between G-quadruplex structures and ribosome footprint density. This analysis revealed the presence of very low ribosome footprint density in the vicinity of the G-quadruplex structures. Further I have analysed the effect of the pharmacological stabilisation of the G-quadruplex on translation using PhenDC3 drug. The treatment of PhenDC3 drug was found to have no significant effect on the translational level. This is described in chapter 2. The presence of ribosome pauses in the upstream of G-quadruplex regions and the lack of an existing tool to identify ribosome pause sites motivated me to develop a tool for the prediction of the ribosome pauses using ribosome profiling data. This lead to the development of PausePred tool which has been discussed in chapter 3. The PausePred tool can be used to infer ribosome pauses which are scored based on their magnitude relative to the background density within the surrounding area. In addition to the score, PausePred provides the coordinates of the pause location, the footprint density at the pause site and the flanking nucleotide sequence. Further I utilized PausePred to analyse ribosome pausing using ribosome profiling data obtained under varying concentrations of polyamines (described in chapter 4). I showed that in cells with higher polyamine levels, proline-proline or proline-glycine amino acids are enriched in the E and P-sites of paused ribosomes.
G-quadruplex , Translation , Ribosome stalling , Ribo-seq , PhenDC3 , RpS6 phosphorylation , Reverse transcriptase slippage , Ribosome profiling , Ribosome pause
Kumari, R. 2018. RNA G-quadruplexes and polyamines effects on translation: data analysis and development of software for ribosome pause prediction. PhD Thesis, University College Cork.