The mRNA cap epitranscriptome: Understanding an essential novel layer of gene expression in neuronal differentiation and function
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
The information for life is encoded in the DNA of the genes harboured in 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 into a messenger RNA intermediate (mRNA) that has a cap structure and a polyA tail to protect it from degradation. This mRNA is then translated in the cytoplasm into a chain of amino acids called proteins, which fulfil 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 the sequence of mRNA only consist of four nucleotides, many can be modified by addition of small chemical groups to increase the regulatory portfolio and coding capacity. The most prominent modification in mRNA are methyl groups added to the nucleotides adjacent to the cap structure. Animals including humans have two cap methyltransferase enzymes (CMTrs) that add these modifications. Also many parasites have a CMTr gene in their genome that is required for their propagation. In mice, CMTrs are essential and required for neuronal development, however, the biological functions of CMTrs and the mRNA cap modifications remain largely unexplained.
We recently discovered that mutant Drosophila lacking both CMTrs are viable, although they suffer from neurological and learning defects. Intriguingly, we further discovered that in these mutant flies, mRNAs were not properly transported to synapses, which are the sites where signals are transmitted to neighbouring neurons. In particular, we could show that some mRNAs are only made into protein at synapses. Hence, the cap modifications have an essential role in directing the synthesis of new proteins locally at synapses suggesting that this process is required for learning of new associations, that are then stored as memory in the brain. However, we currently do not know which genes are expressed in this way at synapses nor what the sequence code is to direct mRNAs to synapses for localized expression.
We now have the ideal animal model to address the very fundamental questions about how this enigmatic modifications direct local expression of genes to synapses. Our preliminary data indicate that the cap modifications vary between different animals and conditions. Since CMTrs also localize to synapses, our data suggest a dynamic code important for local protein synthesis. In a first step to crack this code, we will identify specific mRNAs that localize to synapses allowing us to build a reporter system to test the code. To complement this analysis we will further determine the sequence preferences of CMTrs in biochemical assays and identify proteins important for CMTr specificity and decoding of the cap modification code.
These studies are essential to understand the vital function of the cap modifications in the regulation of gene expression and how its aberrant regulation can lead to neurological defects in humans, or can be exploited to interfere with viral replication such as in SARS-CoV-2.
Although the sequence of mRNA only consist of four nucleotides, many can be modified by addition of small chemical groups to increase the regulatory portfolio and coding capacity. The most prominent modification in mRNA are methyl groups added to the nucleotides adjacent to the cap structure. Animals including humans have two cap methyltransferase enzymes (CMTrs) that add these modifications. Also many parasites have a CMTr gene in their genome that is required for their propagation. In mice, CMTrs are essential and required for neuronal development, however, the biological functions of CMTrs and the mRNA cap modifications remain largely unexplained.
We recently discovered that mutant Drosophila lacking both CMTrs are viable, although they suffer from neurological and learning defects. Intriguingly, we further discovered that in these mutant flies, mRNAs were not properly transported to synapses, which are the sites where signals are transmitted to neighbouring neurons. In particular, we could show that some mRNAs are only made into protein at synapses. Hence, the cap modifications have an essential role in directing the synthesis of new proteins locally at synapses suggesting that this process is required for learning of new associations, that are then stored as memory in the brain. However, we currently do not know which genes are expressed in this way at synapses nor what the sequence code is to direct mRNAs to synapses for localized expression.
We now have the ideal animal model to address the very fundamental questions about how this enigmatic modifications direct local expression of genes to synapses. Our preliminary data indicate that the cap modifications vary between different animals and conditions. Since CMTrs also localize to synapses, our data suggest a dynamic code important for local protein synthesis. In a first step to crack this code, we will identify specific mRNAs that localize to synapses allowing us to build a reporter system to test the code. To complement this analysis we will further determine the sequence preferences of CMTrs in biochemical assays and identify proteins important for CMTr specificity and decoding of the cap modification code.
These studies are essential to understand the vital function of the cap modifications in the regulation of gene expression and how its aberrant regulation can lead to neurological defects in humans, or can be exploited to interfere with viral replication such as in SARS-CoV-2.
Technical Summary
Cap-adjacent nucleotides in mRNA can be dynamically methylated at the 2` position of the ribose (cOMe). This epitranscriptomic code of cOMe is written by cap methyl transferases (CMTrs). Animals generally have two broadly expressed CMTrs, but many viruses also encode one CMTr in their genome, which is required for their propagation. In mice, cOMe is essential and required for neuronal development. The biological functions of cOMe, however, remain largely unknown.
We have adopted Drosophila as the first genetically tractable animal model to reveal functions for cOMe by double knock-out of the two CMTr genes. Flies lacking cOMe have neurological defects including impaired reward learning. Importantly, we show that cOMe is required for localization of untranslated mRNAs to synapses and local protein synthesis. These breakthroughs demonstrate that Drosophila is an excellent model to study the biological functions of mRNA cap methylation and its impact on gene expression.
This proposal will capitalize on the advantage of having a viable cOMe devoid animal model to elucidate how cOMe is used to provide a yet to be characterised essential layer of gene expression control. To elucidate the cap epicode, we will determine the complement of synapse localizing mRNAs to identify common sequence motifs and how they direct installation of cOMe by CMTrs in biochemical assays. Through this approach we will be able to build a reporter system to test the code. Moreover, we will determine CMTr interacting proteins and cOMe readers to build the molecular framework for cOMe installation and decoding.
This study will be the first to provide fundamental insights into how the dynamics of cOMe epimarks is used to direct local protein synthesis at synapses. Moreover, the heterogeneity of transcription start sites in combination with variable methylation suggest a yet to be described major layer of gene expression control with fundamental implications for health and disease.
We have adopted Drosophila as the first genetically tractable animal model to reveal functions for cOMe by double knock-out of the two CMTr genes. Flies lacking cOMe have neurological defects including impaired reward learning. Importantly, we show that cOMe is required for localization of untranslated mRNAs to synapses and local protein synthesis. These breakthroughs demonstrate that Drosophila is an excellent model to study the biological functions of mRNA cap methylation and its impact on gene expression.
This proposal will capitalize on the advantage of having a viable cOMe devoid animal model to elucidate how cOMe is used to provide a yet to be characterised essential layer of gene expression control. To elucidate the cap epicode, we will determine the complement of synapse localizing mRNAs to identify common sequence motifs and how they direct installation of cOMe by CMTrs in biochemical assays. Through this approach we will be able to build a reporter system to test the code. Moreover, we will determine CMTr interacting proteins and cOMe readers to build the molecular framework for cOMe installation and decoding.
This study will be the first to provide fundamental insights into how the dynamics of cOMe epimarks is used to direct local protein synthesis at synapses. Moreover, the heterogeneity of transcription start sites in combination with variable methylation suggest a yet to be described major layer of gene expression control with fundamental implications for health and disease.
