Towards determining the role of m6A-modified LINE-1 in Human Brain Development

Abstract

LINE1s are a subfamily of retrotransposons occupying around 17-21% of our genome. Most of these have lost their ability to transpose on their own, due to truncation of 5’UTR or the accumulation of mutations. Despite this, they can act as cis-regulatory elements and affect gene expression. One RNA modification has been proposed to regulate their expression is called N6-methyladenosine (m6A). It is the most common post-translational RNA modification deposited by a writer complex (METTL3, METTL14, and other proteins) and removed by erasers (e.g. ALKBH5). Both LINE1 expression and the m6A RNA modification are highly prevalent in the human brain where they have been proposed to play a role in human brain development.

This study aimed to investigate whether LINE1s are modified by the m6A RNA modification in human neural cells and to characterize if and how m6A-modified LINE1s affect the host transcriptome and cellular identity of human neural cells.

In the present work, we found that CRISPRi efficiently silences METTL3 in human neuroepithelial-like stem cells but that loss of METTL3 may affect the expression of neural markers and the cells’ ability to differentiate into neurons. We further optimized an RNA immunoprecipitation method for locating m6A-modified LINE1s (meRIP) and designed plasmids for targeted m6A demethylation to study how this modification is distributed on LINE1s and whether these elements influence the host transcriptome. To summarize, the work of this thesis has contributed to the optimization of several methods that will bring us closer towards determining the role of m6A-modified LINE1s in human neural cells.

Popular Abstract

Towards determining the role of m6A-modified LINE1s in Human Brain Development

Transposable Elements also known as “jumping” genes are parts of our genome that can “jump” from one place in our genome to another. One group of them is very common in our genome called LINE1. LINE1s can contribute to genomic diversity and therefore play a role in the development of the complex human brain. At the same time, they can contribute to various neurological disorders and cancer. Therefore, it could be of great importance to study how LINE1s are regulated in our brain, both to better understand the healthy human brain and to better understand mechanisms that, when lost, can lead to disease. Our organisms had to develop mechanisms that would control them. One of these mechanisms is called the m6A RNA modification. Both LINE1 and m6A have been proposed to play a role in brain development. This leaves us with a question of how m6A controls LINE1 and how it affects our brain development.

To answer this question, we removed one of the proteins that are responsible for depositing m6A, called METTL3 in human neural cells, which are cells that are not yet neurons but have the potential to become neurons. We wanted to observe how the removal of METTL3 would affect the cells. What we found out was that cells without METTL3 were not able to become neurons.

But to know how m6A controls LINE1, we also need to ask if are LINE1s m6A-modified and if so then where. So, we decided to try to localise it with a method called MeRIP, which is a method that would find where m6A is in our transcriptome. It works by first chopping up the RNA into small pieces, then we add a protein (an antibody) that looks for the m6A modification that sits on those small pieces, captures them and pulls them away from fragments that are free from the modification. Allowing us to collect fragments that contain the modification. We can then study which LINE1s that are m6A modified and how they affect gene expression. We have never performed this method, so we had to perform trial runs and make sure it worked. Once we confirmed the effectiveness of the trial runs we performed MeRIP on samples where METTL3 had been removed and then sent them later for sequencing that will answer our question about the deposition of m6A modification on LINE1s

Once we receive our results from sequencing, which will narrow the search for m6A-modified LINE1, we plan to sequence with different sequencing methods and compare them. Once we find m6A-modified LINE1s we plan to remove the modification from them to see how it will affect neural cells and their ability to change to neurons.

To summarize, the work of this thesis has contributed to the optimization of several methods that will bring us closer towards determining the role of m6A-modified LINE1s in human neural cells.

Master’s Degree Project in Molecular Biology 60credits 2024
Department of Biology, Lund University
Advisor: Dr Johan Jakobsson
Laboratory of Molecular Neurogenetics, Faculty of Medicine, Lund University

• author: Czeladzka, Marta
• supervisor: Johan Jakobsson
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20232
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9176124
• date added to LUP: 2024-10-04 15:14:53
• date last changed: 2024-10-04 15:14:53