Reconstitution of the 3’-end cleavage reaction reveals Mpe1 in yeast and RBBP6 in humans are critical for activation of CPF and CPSF protein complexes respectively
CryoEM structure of the polymerase module of CPF in complex with RNA, the PSR of Mpe1 and the yPIM of Cft2. The structure shows how Mpe1 (orange) directly contacts RNA (gray), and Cft2 (light blue) binds to the polymerase module of the yeast cleavage and polyadenylation factor (CPF).
Maturation of the 3ʹ-end of messenger RNAs (mRNAs) is a key step of gene expression. This is performed by a multi-subunit protein complex named the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). 3ʹ-end processing requires an endonucleolytic cleavage of the nascent pre-mRNA, followed by the addition of a poly(A) tail at its free 3’-end. The cleavage event defines the end of mature mRNAs, which can dictate mRNA localisation, translation and stability. Activation of CPSF/CPF is tightly regulated to control where cleavage occurs. On its own, CPSF/CPF is an inactive endonuclease and exactly how it is activated has long been an outstanding question. Two new papers from Lori Passmore’s group in the LMB’s Structural Studies Division seek to provide some clarity on this issue.
Specifically, the group have found that the yeast protein Mpe1, and its human orthologue RBBP6, are critical for the correct activation of yeast CPF and human CPSF respectively. In a Genes & Development paper, PhD student Vytaute Boreikaite used recombinant proteins to make the first reconstitution of the 3’-end cleavage reaction in humans, identifying the importance of RBBP6.
In a second Molecular Cell paper, Juan Rodríguez-Molina, another member of Lori’s group, determined the structures of subcomplexes of yeast CPF by electron cryo-microscopy (cryo-EM), finding that Mpe1 directly contacts the polyadenylation signal sequence in the bound mRNA. Mpe1 uses highly conserved residues to interact with RNA and other proteins of CPF, which the Genes & Development paper also identified as critical for the activation of human CPSF by RBBP6 mediation.
This research answers a decades old question on whether human RBBP6 and yeast Mpe1 have a functionally conserved role in 3’-end processing. The potential involvement of human RBBP6 was overlooked for a period because, unlike Mpe1 which is a stable subunit of yeast CPF, RBBP6 is not stably associated with human CPSF. By reconstituting the human 3’-end processing reaction in a test tube, Vytaute showed that RBBP6 is in fact essential for the activation of human CPSF. The structural insights from Juan’s experiments show how Mpe1, and possibly RBBP6, is able to directly contact RNA and CPF to mediate 3’-end processing.
These studies highlight how using model organisms, such as yeast used by Juan, to study highly conserved processes can help us to understand how fundamental processes occur in all cells. The papers show the success of reconstituting complex biological processes in detail within test tubes with fully purified components as a method of understanding biological processes from first principles.
Moreover, this greater understanding of 3’-end processing holds important clinical implications as compounds that inhibit the human 3’-end processing endonuclease have been shown to have anti-cancer and anti-inflammatory properties. Lori’s group found that at least one such compound inhibits the human 3’ endonuclease reaction in a test tube and thus their newly reconstituted human 3’-end processing system could be further developed for drug screening purposes.
The work was funded by UKRI MRC, European Research Council, Herchel Smith Fund, the Wellcome Trust. The authors also acknowledge Diamond Light Source for access to eBIC.
RBBP6 activates the pre-mRNA 3’-end processing machinery in humans. Boreikaite, V., Elliott, TS., Chin, JW., Passmore, LA. Genes & Development
Mpe1 senses the binding of pre-mRNA and control 3’-end processing by CPF. Rodríguez-Molina, JB., O’Reilly, FJ., Fagarasan, H., Sheekey, E., Maslen, S., Skehel, JM., Rappsilber, J., Passmore, LA. Molecular Cell
Lori Passmore’s group page
Jason Chin’s group page