Final Report Summary - POLYAMECHANISMS (Macromolecular machines that regulate mRNA poly(A) tails: mechanisms of polyadenylation and deadenylation)
My laboratory aims to understand the molecular mechanisms of multi-protein complexes with an emphasis on the macromolecular machines that add or remove mRNA polyA tails. We are establishing how polyA tail position and length are determined by the activities of Cleavage and Polyadenylation Factor (CPF), Pan2–Pan3 and Ccr4–Not. These complexes regulate mRNA stability and the efficiency of translation, playing important roles in regulating gene expression.
Over the course of this ERC Starting grant PolyAMechanisms, we established methods to reconstitute CPF, Pan2-Pan3 and Ccr4-Not complexes, and have studied them using structural, biochemical and functional techniques. This led to new insights into the link between transcription and polyadenylation. Specifically, we showed that the Glc7 phosphatase of CPF plays a key role in transcription termination by dephosphorylating phospho-Tyr1 of the C-terminal domain of the largest subunit of RNA polymerase II. This provides a clear mechanistic link between mRNA 3’ end processing and transcriptional regulation.
We also reconstituted the two major deadenylation complexes in eukaryotes – Pan2-Pan3 and Ccr4-Not, leading to new understanding of their molecular mechanisms. Using fully recombinant proteins, we show that Pan3 is required to recruit RNA to the catalytic Pan2 subunit. Crystal and NMR structures provide insight into how the complex assembles and functions. By reconstituting the seven-subunit, 500 kDa Ccr4-Not complex we show that it has an intrinsic preference for single stranded RNAs. We also identified a novel RNA-binding adapter protein for Ccr4-Not from the fission yeast, S. pombe. We show using in vitro assays that this protein, Mmi1, accelerates deadenylation by Ccr4-Not on target RNAs.
Additionally, we investigated methods for electron cryo-microscopy (cryo-EM). We developed new substrates that improve image quality, enabling structure determination of more difficult proteins than was previously possible. We showed that partial hydrogenation of graphene supports allows tunable and reproducible protein adsoption onto the surface. We also developed ultrastable gold supports that are now in routine use in the EM community and are commercially available. These improved supports reduce radiation-induced blurring of images, improving the structures of proteins imaged on them.
Over the course of this ERC Starting grant PolyAMechanisms, we established methods to reconstitute CPF, Pan2-Pan3 and Ccr4-Not complexes, and have studied them using structural, biochemical and functional techniques. This led to new insights into the link between transcription and polyadenylation. Specifically, we showed that the Glc7 phosphatase of CPF plays a key role in transcription termination by dephosphorylating phospho-Tyr1 of the C-terminal domain of the largest subunit of RNA polymerase II. This provides a clear mechanistic link between mRNA 3’ end processing and transcriptional regulation.
We also reconstituted the two major deadenylation complexes in eukaryotes – Pan2-Pan3 and Ccr4-Not, leading to new understanding of their molecular mechanisms. Using fully recombinant proteins, we show that Pan3 is required to recruit RNA to the catalytic Pan2 subunit. Crystal and NMR structures provide insight into how the complex assembles and functions. By reconstituting the seven-subunit, 500 kDa Ccr4-Not complex we show that it has an intrinsic preference for single stranded RNAs. We also identified a novel RNA-binding adapter protein for Ccr4-Not from the fission yeast, S. pombe. We show using in vitro assays that this protein, Mmi1, accelerates deadenylation by Ccr4-Not on target RNAs.
Additionally, we investigated methods for electron cryo-microscopy (cryo-EM). We developed new substrates that improve image quality, enabling structure determination of more difficult proteins than was previously possible. We showed that partial hydrogenation of graphene supports allows tunable and reproducible protein adsoption onto the surface. We also developed ultrastable gold supports that are now in routine use in the EM community and are commercially available. These improved supports reduce radiation-induced blurring of images, improving the structures of proteins imaged on them.