The impact and regulation of eIF4A-multimerisation in establishing translational programmes
Lead Research Organisation:
University of Glasgow
Department Name: College of Medical, Veterinary, Life Sci
Abstract
Genes are the blueprints for building an organism and define its biological properties. They are made of DNA that is kept in the nucleus of the cell. In the process of gene expression, to create what is encoded by a gene, DNA is copied and made in a new form as RNA. The information within RNAs is read by molecular machines, called ribosomes, that translate and convert this information into amino acid sequences named proteins. The collection of proteins within a cell is termed the proteome.
RNA and the proteome are the main particles to carry out functions within cells. Altering the RNAs within a cell enables dynamic changes to the proteome and, hence, changes cell function and fate. Groups of genes are co-ordinately regulated and are combined to form a programme of cell activity that dictate which RNAs produce which proteins at specific times. Unfortunately, these mechanisms can be faulty due to mutations in genes that regulate the expression of these genes. Such dysregulation can lead to activation of cellular programmes at the wrong time producing RNA and proteins that cause fatal illnesses. A devastating example is cancer, when RNAs lead to uncontrolled production of proteins related to cell division.
Most cellular processes that dictate which RNAs are selected for translation into protein operate by adjusting the function of the translation initiation complex eIF4F. At its hearts operates a protein called eIF4A1. This protein is essential for loading RNAs onto ribosomes and starting protein translation. eIF4A1 activity is changed by interacting with many other proteins, called cofactors. In addition, RNAs themselves can contain elements that may change which function of eIF4A1 is needed. Researchers have gathered information on how eIF4A1 activity is changed by its cofactors and are starting to understand that dysregulation of eIF4A1 has fatal outcomes. However, we still do not know why and how this dysregulation is established. To shed light on this, we first need to know how eIF4A1 function is controlled by its cofactors and RNA targets which together dictate which of these RNAs are selected for translation by the ribosome. This research proposal aims to identify these patterns and mechanisms of eIF4A1 regulation.
It has been believed that eIF4A1 is active as a single molecule but our recent work revealed that eIF4A1 forms protein complexes that are assembled from one or three copies of it. We also gathered evidence that these complexes have different activities and that RNA affects the distribution between these different states. This creates the hypothesis of how RNA might dictate eIF4A1 activity. Thus, this research will focus particularly on the power of RNA itself to direct activity of eIF4A1.
To achieve this goal, we need to understand which RNAs recruit which eIF4A1 complex and exactly define the eIF4A1 activity required by the RNA targets. To do this, we will isolate the RNAs that are specifically bound by different eIF4A1 complexes. We will then use computational tools to find out what discriminates these RNAs from others. To understand how the multi-eIF4A1 complexes perform their function on RNA we will determine the 3D structure of the complexes using state-of-the-art microscopes. This will identify how the complexes form and enable us to generate modified eIF4A1 that cannot form these complexes. We will use the modified complexes to dissect their activities and reconstitute key steps of how RNAs are translated into protein in a cell-free environment to understand their function. Together these findings will identify a relationship between eIF4A1, RNAs and cofactors that ultimately dictate protein translation. With this, we will contribute fundamental knowledge to increase our understanding of the regulation and dysregulation of eIF4A1 and how this affects cell function. In the further it may allow specific drugs to be designed that will affect specific eIF4A1 activities.
RNA and the proteome are the main particles to carry out functions within cells. Altering the RNAs within a cell enables dynamic changes to the proteome and, hence, changes cell function and fate. Groups of genes are co-ordinately regulated and are combined to form a programme of cell activity that dictate which RNAs produce which proteins at specific times. Unfortunately, these mechanisms can be faulty due to mutations in genes that regulate the expression of these genes. Such dysregulation can lead to activation of cellular programmes at the wrong time producing RNA and proteins that cause fatal illnesses. A devastating example is cancer, when RNAs lead to uncontrolled production of proteins related to cell division.
Most cellular processes that dictate which RNAs are selected for translation into protein operate by adjusting the function of the translation initiation complex eIF4F. At its hearts operates a protein called eIF4A1. This protein is essential for loading RNAs onto ribosomes and starting protein translation. eIF4A1 activity is changed by interacting with many other proteins, called cofactors. In addition, RNAs themselves can contain elements that may change which function of eIF4A1 is needed. Researchers have gathered information on how eIF4A1 activity is changed by its cofactors and are starting to understand that dysregulation of eIF4A1 has fatal outcomes. However, we still do not know why and how this dysregulation is established. To shed light on this, we first need to know how eIF4A1 function is controlled by its cofactors and RNA targets which together dictate which of these RNAs are selected for translation by the ribosome. This research proposal aims to identify these patterns and mechanisms of eIF4A1 regulation.
It has been believed that eIF4A1 is active as a single molecule but our recent work revealed that eIF4A1 forms protein complexes that are assembled from one or three copies of it. We also gathered evidence that these complexes have different activities and that RNA affects the distribution between these different states. This creates the hypothesis of how RNA might dictate eIF4A1 activity. Thus, this research will focus particularly on the power of RNA itself to direct activity of eIF4A1.
To achieve this goal, we need to understand which RNAs recruit which eIF4A1 complex and exactly define the eIF4A1 activity required by the RNA targets. To do this, we will isolate the RNAs that are specifically bound by different eIF4A1 complexes. We will then use computational tools to find out what discriminates these RNAs from others. To understand how the multi-eIF4A1 complexes perform their function on RNA we will determine the 3D structure of the complexes using state-of-the-art microscopes. This will identify how the complexes form and enable us to generate modified eIF4A1 that cannot form these complexes. We will use the modified complexes to dissect their activities and reconstitute key steps of how RNAs are translated into protein in a cell-free environment to understand their function. Together these findings will identify a relationship between eIF4A1, RNAs and cofactors that ultimately dictate protein translation. With this, we will contribute fundamental knowledge to increase our understanding of the regulation and dysregulation of eIF4A1 and how this affects cell function. In the further it may allow specific drugs to be designed that will affect specific eIF4A1 activities.
Technical Summary
eIF4A1 is a DEAD-box RNA helicase operating as the heart of the translation initiation complex eIF4F. Distinct enzymatic activities of eIF4A1 are required to perform its functions in loading mRNAs into the pre-initiation complex (PIC) and resolving RNA structure to allow PIC scanning. Different mRNAs require different combinations of these eIF4A1 activities to facilitate their optimal translation initiation. We discovered novel, multimeric eIF4A1 complexes that form specifically on certain RNA sequences and that are required for efficient RNA unwinding. This proposal aims to comprehensively investigate how multimerisation of eIF4A1 regulates its biological functions by the formation of distinct complexes.
We will globally identify the mRNA targets that are differentially dependent on eIF4A1 activities using ribosome profiling with specific inhibitors which distinctly affect the monomeric and multimeric states of eIF4A1. This will be coupled with the determination of which eIF4A1-cofactors contribute to the translation of specific groups of mRNAs. In parallel, in vitro DART-seq and multimerisation-mutants of eIF4A1 will be used to deconvolute the mRNA-specific activities of monomeric and multimeric eIF4A1 in mRNA loading and scanning. Data analysis using machine learning will generate an unparalleled view of eIF4A1's catalytic relationship to its mRNA targets. Results will be confirmed with biochemical assays.
To understand the assembly and the mechanism of unwinding of the multimeric eIF4A1 complexes, we will determine their structure. We will then use this information to generate multimerisation-defective eIF4A1 mutants and examine their functional defects.
To determine if helicase-multimerisation is critical for all DEAD-box RNA helicases, we will take emerging key features and apply them to eIF4A1-related helicases. RNA specificities of these helicases will be determined and the relationship between multimerisation and catalytic activities examined in vitro
We will globally identify the mRNA targets that are differentially dependent on eIF4A1 activities using ribosome profiling with specific inhibitors which distinctly affect the monomeric and multimeric states of eIF4A1. This will be coupled with the determination of which eIF4A1-cofactors contribute to the translation of specific groups of mRNAs. In parallel, in vitro DART-seq and multimerisation-mutants of eIF4A1 will be used to deconvolute the mRNA-specific activities of monomeric and multimeric eIF4A1 in mRNA loading and scanning. Data analysis using machine learning will generate an unparalleled view of eIF4A1's catalytic relationship to its mRNA targets. Results will be confirmed with biochemical assays.
To understand the assembly and the mechanism of unwinding of the multimeric eIF4A1 complexes, we will determine their structure. We will then use this information to generate multimerisation-defective eIF4A1 mutants and examine their functional defects.
To determine if helicase-multimerisation is critical for all DEAD-box RNA helicases, we will take emerging key features and apply them to eIF4A1-related helicases. RNA specificities of these helicases will be determined and the relationship between multimerisation and catalytic activities examined in vitro