Periodic Reporting for period 4 - POLYAMACHINES (The polyA machinery: Elucidating the molecular mechanisms of mRNA polyadenylation, deadenylation and RNA recognition)
Reporting period: 2021-10-01 to 2022-12-31
Our genetic code is translated from DNA into proteins through an intermediate molecule, messenger RNA (mRNA). Controlling the amount of mRNA produced, its lifetime, and how efficiently it is translated into protein regulates the amount of protein produced in the cell. All of these processes can be tightly controlled to allow rapid responses to cellular stimuli.
In eukaryotes, a tail of adenosines at the 3’ end of the mRNA (the poly(A) tail) contributes to the control of mRNA stability and the efficiency of translation. Poly(A) tails are added by the Cleavage and Polyadenylation Factor (CPF/CPSF) and removed by the Ccr4–Not and Pan2–Pan3 multiprotein complexes. These complexes control expression of genes in the inflammatory response, in stress responses, and during oocyte development, for example. They are deregulated in disease, including cancer, viral infection and neurological disorders.
Although many of the proteins that add and remove poly(A) tails were known, their mechanisms were poorly understood. In this proposal, my objective was to understand the molecular basis for addition and removal of poly(A) tails on specific mRNAs. We determined structures of poly(A) tail regulatory complexes, studied their activities through biochemical reconstitution, and performed functional studies in cells. Through this integrated approach, we determined the overall architecture of CPF and showed that its four enzymatic activities are contained in three different modules. We also reconstituted specific and efficient pre-mRNA cleavage for the first time from recombinant yeast and human proteins. By determining structures of CPF and performing in vitro studies, we have shown how RNA is recognised and that the complex is highly regulated to prevent spurious cleavage and promote fidelity in mRNA 3’-end processing.
We also provided new insights into the specificity of deadenylases, how deadenylation is coupled to translation, how it is influenced by poly(A) binding protein, and how specific mRNAs are targeted. Together, our work has provided new biological and technological insights, leading to understanding of fundamental processes in gene expression and the role of poly(A) tails.
In eukaryotes, a tail of adenosines at the 3’ end of the mRNA (the poly(A) tail) contributes to the control of mRNA stability and the efficiency of translation. Poly(A) tails are added by the Cleavage and Polyadenylation Factor (CPF/CPSF) and removed by the Ccr4–Not and Pan2–Pan3 multiprotein complexes. These complexes control expression of genes in the inflammatory response, in stress responses, and during oocyte development, for example. They are deregulated in disease, including cancer, viral infection and neurological disorders.
Although many of the proteins that add and remove poly(A) tails were known, their mechanisms were poorly understood. In this proposal, my objective was to understand the molecular basis for addition and removal of poly(A) tails on specific mRNAs. We determined structures of poly(A) tail regulatory complexes, studied their activities through biochemical reconstitution, and performed functional studies in cells. Through this integrated approach, we determined the overall architecture of CPF and showed that its four enzymatic activities are contained in three different modules. We also reconstituted specific and efficient pre-mRNA cleavage for the first time from recombinant yeast and human proteins. By determining structures of CPF and performing in vitro studies, we have shown how RNA is recognised and that the complex is highly regulated to prevent spurious cleavage and promote fidelity in mRNA 3’-end processing.
We also provided new insights into the specificity of deadenylases, how deadenylation is coupled to translation, how it is influenced by poly(A) binding protein, and how specific mRNAs are targeted. Together, our work has provided new biological and technological insights, leading to understanding of fundamental processes in gene expression and the role of poly(A) tails.
Overall, we have published 15 reserach manuscripts and 6 reviews based on POLYAMACHINES so far. Several additional manuscripts are in preparation. We determined structures of poly(A) tail regulatory complexes, studied their activities through biochemical reconstitution, and performed functional studies in cells. Through this integrated approach, we determined the overall architecture of CPF and showed that its four enzymatic activities are contained in three different modules (Casanal et al 2017). We also reconstituted specific and efficient pre-mRNA cleavage for the first time from recombinant yeast and human proteins (Hill et al 2019; Boreikaite et al 2022). By determining structures of CPF and performing in vitro studies, we have shown how RNA is recognised and that the complex is highly regulated to prevent spurious cleavage and promote fidelity in mRNA 3’-end processing (Rodriguez-Molina et al 2022).
We also provided new insights into the specificity of deadenylases, how deadenylation is coupled to translation, how it is influenced by poly(A) binding protein, and how specific mRNAs are targeted (Webster et al 2018; Webster et al 2019). We showed how poly(A) RNA can be recognised indirectly, through its structure rather than through base-specific interactions (Tang et al 2019). Together, our work has provided new biological and technological insights, leading to understanding of fundamental processes in gene expression and the role of poly(A) tails.
We also provided new insights into the specificity of deadenylases, how deadenylation is coupled to translation, how it is influenced by poly(A) binding protein, and how specific mRNAs are targeted (Webster et al 2018; Webster et al 2019). We showed how poly(A) RNA can be recognised indirectly, through its structure rather than through base-specific interactions (Tang et al 2019). Together, our work has provided new biological and technological insights, leading to understanding of fundamental processes in gene expression and the role of poly(A) tails.
We use cutting-edge methods to allow us to overexpress all subunits of megadalton-sized multi-protein complexes using baculoviruses and insect cells. Thus, we are able to obtain milligram quantities of pure protein. In turn, this has allowed us to fully reconstitute parts of mRNA poly(A) tail addition and removal in vitro, to understand the regulation and mechanisms of these processes (Webster et al 2018; Hill et al 2019; Boreikaite et al 2022). For example, we established two-colour assays to evaluate how Ccr4-Not removes poly(A) tails from specific mRNAs in a competitive situation (Webster et al 2019) and to monitor cleavage by CPF (Hill et al 2019).