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The polyA machinery: Elucidating the molecular mechanisms of mRNA polyadenylation, deadenylation and RNA recognition

Periodic Reporting for period 3 - POLYAMACHINES (The polyA machinery: Elucidating the molecular mechanisms of mRNA polyadenylation, deadenylation and RNA recognition)

Reporting period: 2020-04-01 to 2021-09-30

Our genetic code is translated from DNA into proteins through an intermediate molecule, messenger RNA (mRNA). Regulation of the amount of mRNA produced, the lifetime of mRNA, and how efficiently the mRNA is translated into protein all control the final amount of protein 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 or removed by the Cleavage and Polyadenylation Factor (CPF), Ccr4–Not and Pan2–Pan3 multiprotein complexes. They control expression of genes in the inflammatory response, in stress responses, and during oocyte development, for example. These processes are deregulated in disease, including cancer, viral infection and neurological disorders.

Although the proteins that add and remove poly(A) tails are known, their mechanisms are poorly understood. My lab recently established methods to reconstitute the poly(A) tail machinery. This led to new insights into the link between transcription and poly(A) tail addition, new understanding of the molecular mechanisms of poly(A) tail removal, and details of how specific RNAs are regulated.

In this proposal, my objective is to understand the molecular basis for addition and removal of poly(A) tails on specific mRNAs. We will determine high-resolution structures of the poly(A) tail machinery using electron cryo-microscopy (cryoEM), understand their activities through biochemical reconstitution, and study their functional roles in yeast or human cells. We use this integrated approach to study intact multiprotein complexes where we can, although we sometimes investigate individual subunits or domains. This involves considerable technical challenges and an investment in developing high quality purifications and new structural methods. We will determine how the four enzymatic activities of CPF are coupled, the mechanisms by which Ccr4–Not targets specific mRNAs, and the molecular basis for RNA recognition by Pan2–Pan3. Together, this will provide new biological and technological insights, leading to understanding of fundamental processes in gene expression and the role of poly(A) tails in disease.
We have made excellent progress in POLYAMACHINES during this reporting period. We established purification protocols so we are now able to isolate all three multi-protein complexes (CPF, Ccr4-Not and Pan2-Pan3) as well as their cofactors. We also established assays to investigate their activities in vitro.

We determined a high-resolution cryoEM structure for a large part of the CPF (the polymerase module that is required to add poly(A) tails) (Casañal, Kumar et al., Science 2017). We also determined a model for the cleavage and polyadenylation machinery, based on lower resolution structures, biochemical assays and interaction studies (Hill et al., Mol Cell 2019).

We determined a crystal structure of the Pan2 enzyme to reveal how poly(A) RNA binds to the enyzmes that remove poly(A) (Tang et al., NSMB 2019). Surprisingly, Pan2 recognises the distinct shape of the poly(A) RNA rather than recognising its unique chemical features.

We also gained substantial insight into how specific mRNAs are targeted for poly(A) tail removal, and therefore degradation. This work is based on biochemical reconstitutions (Webster et al., Mol Cell 2018; Webster et al., eLIFE 2019).
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., Methods 2018). For example, we established a two-colour assay to evaluate how Ccr4-Not removes poly(A) tails from specific mRNAs in a competitive situation (Webster et al., eLIFE 2019).

In addition, we combine many methods in an integrated approach to obtain molecular insight.

We expect to gain further insights into how the enzymes of CPF are coupled, how CPF binds RNA, and how Ccr4-Not targets specific mRNAs for poly(A) tail removal.