Grant: CIHR Fellowship

Messenger RNAs (mRNA) are copies of genes that contain the information necessary to produce proteins. The formation of the right cell type depends on the production of the correct mRNAs that contain the instructions to make this given cell type. This is achieved in part through RNA splicing, in which certain parts of the premature RNA called introns are removed to form the mature mRNA. Defects in RNA splicing are a common cause of many human disorders, including cancer and neurodegenerative diseases. However, the mechanisms that control splicing in human cells are not well understood, which is mainly due to a lack of tools to study splicing while it is occurring. We recently developed a new method, nanopore analysis of CO-transcriptional Processing (nano-COP), which relies on direct RNA sequencing and allows us to monitor splicing while new RNAs are being produced. For example, we gained insight into how fast splicing occurs and the order of intron removal in human leukemia cells. The objective of this project is to use nano-COP to better understand how these aspects of splicing are controlled in various cell types. We will first apply nano-COP to measure splicing in 10 different human cell lines, including normal and cancer cells, to determine how splicing dynamics vary across cell types and in cancer. Next, we will use white blood cells from 10 different individuals to examine how splicing kinetics and order are impacted by genetic variants in parts of the RNA that are known to cause abnormal splicing. Finally, we will remove three proteins that play a role in splicing to determine in which aspect of splicing they are most important. We will repeat this experiment in three different cell types to discover whether certain functions of these proteins are cell type-specific. This study will improve our understanding of the control of the splicing process, which is crucial in order to learn how it goes awry in various human diseases.