The impact of post-transcriptional RNA processing on macrophage response to exogenous stimulation

Supervisors: Prof. Bruce Whitelaw, Dr Musa Hassan, Prof. Eileen Wall

Project description:

Our own work [1] and independent studies [2] have shown that genetic variants that affect RNA processing: 1) are abundant, 2) occur independently of those that affect gene expression, and 3) are linked to a variety of complex traits in humans and animals. A full understanding of the molecular mechanisms through which these variants contribute to phenotypic diversity – particularly in livestock – is however, still lacking. This project seeks to quantitatively assess genome-wide transcript abundance, isoform usage, splicing, and mRNA translation by applying a recent protocol that couples template-switching PCR with the depth and breadth of next generation RNA sequencing to profile mRNAs that are occupied by ribosomes (an indicator of translation) from low input data [3].

Aim 1 will elucidate the impact of variable RNA processing on bovine macrophage response to IFNG stimulation or infection with Toxoplasma or Neospora. It will also include maturation of the bioinformatics pipeline to analyze resulting sequence data.

Aim 2 will decipher the parasite’s response to changes in the host cell microenvironment. It will exploit dual RNA- and Ribo-seq to reveal Toxoplasma and Neospora transcriptional and translational reprogramming in naïve and IFNG-stimulated macrophages.

The proposed work will yield a comprehensive view of the role for RNA processing in the host and pathogen, as well as new information on the extent of translation of individual gene products. In addition, it will reveal the functions of the numerous recently identified short open-reading frames (sORFs) in cattle, Toxoplasma and Neospora.

Through this project, the student should acquire a solid understanding of molecular biology, immunology, and computational biology. He/she will become proficient in a wide variety of cell and molecular biology techniques including derivation, culture and differentiation of mammalian primary cells, western blotting, ribosome profiling, genome editing, and bioinformatics. The student will also acquire skills in communication, writing and project management. The project, brings together the complementary expertise of the co-supervisors Professor Bruce Whitelaw (genetically engineered livestock including gene editing), Dr. Musa Hassan (quantitative genetics and genomics, host-parasite interactions, and computational biology), and Eileen Wall (Genetic breeding for improved animal production), in a new collaboration, where the student will have access to state of the art facilities and resources.