m6A mRNA methylation - understanding an essential mechanism adjusting gene expression during development and differentiation

The information for life is encoded in the DNA of the genes harbored on our chromosomes. The DNA in a chromosome is a very long chain consisting of four different nucleotides: G, A, C and T. For most genes this code is then converted via a messenger RNA intermediate (mRNA) into a chain of amino acids called proteins, which fulfill a function; for example an enzymatic reaction to generate energy from the nutrients we eat to allow for the electrical communication among neurons in our brain.

Although proteins consist of long uninterrupted chains of amino acids, this is not the case for the genetic code in the DNA. The coding sequence of almost all genes in the DNA termed exons is interrupted by non-coding sequences called introns. When the DNA sequence is transcribed into a complementary pre-mRNA the intervening intronic sequences need to be spliced out to make the mature mRNA. Thus, correct splicing of pre-mRNAs is a very important process, but the sequences directing splicing of small exons, which are generally hidden in very large introns are very short and appear fuzzy to us humans. Nevertheless the splicosome cuts introns out very accurately. But there is much more to splicing than simply producing a "correct" transcript, because the exon content can be varied by a process called alternative splicing. Strikingly, it is the amount of alternative splicing in our genes, and not the number of genes that distinguishes us from simpler organisms. These differences are most prominently manifested in the genes that are expressed in our most complex organ, the brain.

Intriguingly, the sequence of RNA does not just consist of four nucleotides, because many can be modified by addition of small chemical groups. The most prominent modification in mRNA is methylation of adenosines (m6A), which is required for viability of mice and plants. We recently discovered that mutant Drosophila lacking the m6A modification are viable, although with neurological and metabolic defects. We further discovered that female viability is compromised. Using sensitive genetic interactions possible due the viability of these mutants allowed us then to reveal a fundamental role of m6A in alternative splicing of the master regulator of sex determination Sex lethal (Sxl) in the fruit fly Drosophila. Sxl has an additional role and is required to adjust gene expression of the unequal numbers of X-chromosomes between males and female; a process called dosage compensation that is most affected in m6A devoid females.
Hence, we now have the ideal animal model to address the very fundamental questions about how this enigmatic modification adjusts expression of genes. Our preliminary data indicate that the m6A modification has important functions in early embryogenesis, when sexual fate is established and the genome is reprogrammed for later development. Accordingly, we will globally map m6A sites in early embryogenesis and use the genetically sensitive Sxl paradigm to ask the very fundamental question how the dynamics of m6A levels are used to adjust gene expression during development and differentiation of cells.
These studies are essential to understand the vital function of the m6A modification in the regulation of gene expression and how its aberrant regulation can lead to neurological and metabolic defects in humans, or can be exploited to interfere with viral replication such as in Zika virus.

Adenosines in mRNA can be dynamically modified by methylation (m6A). This epitranscriptomic code of m6A is written by the methylosome, a large multimeric protein complex, and read by YTH domain proteins, but can also be erased by demethylases. The m6A modification is essential from yeast to man with roles in stem cell biology, human X chromosome inactivation and circadian rhythms, but has also been linked to obesity and neurological disorders through the disease risk locus FTO, which encodes an m6A demethylase.

We have adopted Drosophila as the first genetically tractable animal model to reveal functions for m6A. Flies lacking m6A have neurological defects, but m6A is also required for sex determination and dosage compensation. Importantly, we show that m6A is required for alternative splicing regulation of the master sex determination factor Sex-lethal (Sxl). These breakthroughs demonstrate that Drosophila is an excellent model to study the biological functions of mRNA methylation and its impact on gene expression.

This proposal will capitalize on the advantage of having a viable m6A devoid animal model to elucidate how m6A is used globally during reprogramming of the embryonic genome in early embryogenesis and how m6A contributes to establish stable sexual fates and maintain them during differentiation. In Drosophila, the key step to female differentiation is expression of Sxl through an auto-regulatory loop. We will address how m6A contributes to establish sex-specific gene expression by examining the dynamics of m6A during development and by interrogating the requirement of individual sites for Sxl auto-regulation. These experiments will further reveal if m6A levels are adjusted and epigenetically inherited.

This study will be the first to provide fundamental insights into how the dynamics of m6A epimarks is used to establish stable sexual fate by adopting alternative splicing regulation to adjust gene expression during development and differentiation.

DISEASE AND HEALTH CARE: The role of DNA methylation in cancer and numerous other diseases is the subject of some 30,000 research papers. In contrast, mRNA methylation has, since its discovery over 40 years ago, been rather neglected until recently, when we and others have established RNA methylation as a fundamental mechanism of regulation of gene expression and splicing. Thus, it is to be anticipated that studying m6A methylation of mRNA will have a broad impact on health and wealth in society.

Genome information of individual patients will soon be available for diagnostic purposes and personalized therapies in our health care system. However, for most of the non-coding regions of the genome we are as yet unable to assign regulatory functions. Regulatory sequences involved in alternative mRNA processing, due to their degeneracy and redundancy, typically resist in silico determination of which proteins bind to them or which RNA-based processes they regulate.

In order for health care professionals to exploit genome information more fully, it is imperative to understand mRNA processing codes among non-coding sequences that frequently differ between individuals. Our research will provide major contributions to decipher mRNA processing codes related to alternative mRNA processing regulated by m6A methylation of mRNA. Alternative mRNA processing changes as individuals age and better understanding of this process promises the opportunity for improved diagnostics and potentially new therapeutics leading to major enhancements in this segment of life.

In the long-term elaborating protein-protein and RNA-protein combinatorial interactions promises novel therapeutic approaches to interfere with alternative mRNA processing in a gene-specific fashion. We will actively pursue the exploitation of new data that might potentially underpin future patents. Thus, this proposal is directly relevant to the demand for trained scientists experienced in interpreting genome data. Indeed we will equip trainees with the requisite genetic, bioinformatics and biochemical experience to accelerate advances in this crucial area of research.

BBSRC STRATEGIC PRIORITIES: This proposal encompasses the BBSRC's strategic plan to enhance understanding of the most complex human organ, the brain. Since mRNA processing including mRNA methylation is abundant in the brain, and is altered during aging, the proposed project provides a route for elucidating mechanisms that are risk factors for poor physical and mental health. This project is of particular relevance to the 10-year vision of the BBSRC towards an integrative understanding of organisms as it aims to elucidate regulatory codes at a genomic scale and how these codes operate at an organismal level.

This project also fulfils a strategic aim: "developing and embedding a Systems approach to Biosciences in order to advance fundamental understanding of complex biological processes". Research on alternative mRNA processing has been strongly focused on integrating information from diverse experimental situations. Thus, this project fulfils a BBSRC research priority of "Systems approach to biological research" and implements aims towards predictive biology.

TRAINING: "Providing skilled researchers needed for academic research" is part of BBSRC Strategic aims: We have excellent track records in training and mentoring students and postdoctoral trainees at all levels and provide significant input in laboratory training for individual students as part of University degree programs. This includes graduate students participating in the UoB and UoN BBSRC funded doctoral training programs. This grant proposal promises to help BBSRC achieve this aim, especially as it will disseminate cutting edge technology and high-end quality science such as combining genetic with molecular and biochemical analysis for the interpretation of genomes to set standards for the youngest generation entering careers in research.