Target enrichment of transposable elements for direct RNA sequencing

Co-supervisor: Dr Rebecca Berrens, Department of Paediatrics

Commercial partner: Oxford Nanopore Technologies

Background:

In our conventional understanding, we often assume that all cells in our body possess the same genetic information. However, this is not entirely accurate, as only a small portion of our genome, approximately 2%, comprises protein-coding genes responsible for producing essential functional proteins. Astonishingly, the vast majority of our genome, around 50%, is composed of transposable elements (TEs), which are genetic entities capable of moving and rearranging their positions within the genome.

One particularly important class of transposable elements in humans is called LINE1 (Long Interspersed Nuclear Element 1). LINE1 elements constitute a substantial portion of our genome, accounting for approximately 17%. What sets LINE1 elements apart from other transposable elements is that they have retained their activity over evolutionary time, making them capable of moving around within the genome. As a result of this mobility, about 20% of new mutations in humans can be traced back to LINE1 insertions. Due to such mobility, LINE1 elements have been linked to the development of various congenital diseases, including hemophilia, Duchenne muscular dystrophy, and Dent disease.

Despite our knowledge of the DNA sequence of various LINE1 families, a critical piece of information remains elusive—the precise sequence of a retrotransposition-competent human LINE1 RNA. Retrotransposition refers to the process through which LINE1 elements move to new locations within the genome. Understanding this RNA sequence is of paramount importance for comprehending the role of LINE1 elements in congenital diseases. Therefore, the primary goal of this project is to directly sequence LINE1 RNA molecules, enabling the identification of the exact sequence and any RNA modifications present in a full-length human LINE1 molecule.

Method:

To achieve this ambitious goal, the project focuses on developing a target enrichment method for Nanopore direct RNA-sequencing. This methodology involves creating target enrichment probes designed to specifically capture and isolate the LINE1 RNA of interest. The target enrichment of LINE1 RNA will be done in human embryonic stem cells and human induced pluripotent stem cells, as these cells actively express LINE1 RNA. This project will be done in collaboration with Oxford Nanopore Technologies (ONT). ONT offers the unique possibility of direct RNA sequencing, a method that allows for the analysis of full-length RNA sequences. This technology also enables the mapping of RNA modifications on a single RNA molecule.

The key advantage of using LINE1 RNA for developing a new direct RNA-sequencing enrichment method lies in the fact that LINE1 insertions possess strikingly similar sequences at multiple sites within the genome. This sequence similarity allows for an efficient pulldown of LINE1 RNA using a process called hybridization, wherein the RNA is attracted to and bound by its own matching sequence. Once the target enrichment method is meticulously optimized, it can be applied to perform targeted sequencing of other significant genetic elements, such as single-copy genes or other transposable elements.

Aim:

The goal is to understand the sequence composition of an active LINE1 RNA, to allow the development of small molecule inhibitors to prevent LINE1 de novo insertions that can lead to congenital diseases. These studies could pave our way toward the identification and treatment of diseases with genetic bases.

Apply using course: DPhil in Paediatrics