One of the more interesting aspects of the polyadenylation complex is the nature of the protein-protein interactions that help shape the machinery. A recent study has revealed a fascinating side of this story. It involves the interaction between a subunit known as Fip1 (Fip = Factor Interacting with Poly(A) polymerase) and poly(A) polymerase (affectionately abbreviated as PAP). This study is remarkable for two reasons. For one, it dispels some previously-held notions about the functional significance of this interaction. In addition, it reveals that Fip1 is a member of a large class of proteins that share a common feature – namely, they do not have a rigidly-specified 3-dimensional structure in and of themselves.
This group did two basic things – they solved the crystal structure of PAP in complex with a small part of Fip1, and they studied the solution structure of free Fip1 using a battery of biochemical and biophysical approaches. The structure of the PAP-Fip1 complex is shown here (the illustration taken from the paper by Meinke et al.):
In this illustration, the portion of Fip1 that appears in the structure is shown in red, and PAP is depicted in yellow. What is interesting is that the position of Fip1, as well as several biochemical characterizations that accompanied the description of the structure, indicate that Fip1 likely has no direct effect on the activities (RNA binding, nucleotidyltransferase) of PAP – it does not seem to affect the active site and has at best subtle effects on the overall structure of the protein. This contradicts earlier work performed with the yeast proteins, and also studies of the human and plant Fip1 orthologs. Meinke et al. suggest that Fip1 acts, not a direct effector of PAP activity, but rather as a scaffold that tethers PAP to other of the subunits that Fip1 interacts with (to name three, CPSF30/Yth1, CstF77/Rna14, and, in the case of the human and plant Fip1 orthologs, the RNA substrate itself).
The second point of this study (in my very humble opinion) is that uncomplexed Fip1 exists in solution as a rather unstructured polypeptide. This was determined using a battery of approaches – limited proteolytic digestion, ultracentrifugation, electrophoresis, and CD. The implications of this results are interesting, and may be of importance for far more than just the polyadenylation complex. One of the interesting features of Fip1 is that, of all the polyadenylation complex subunits, it is the least well-conserved at the amino acid sequence level. However, they share some common themes – acidic N-termini, a small but conserved “Fip1 domain”, and, in the case of human and plant proteins, positively-charged domains that are responsible for RNA binding. (It is an acidic domain that interacts with PAP in yeast, animals, and plants.) This implies that the interaction between Fip1 and PAP entails a “molding” of sorts of the unstructured Fip1 polypeptide such that the interaction shown above can occur with reasonable affinity. The fact that the interactions involving Fip1 involve highly-variable parts of the protein suggests a parallel evolutionary flexibility in the interactions involving Fip1, and lends itself to the hypothesis that Fip1 may engage in many lineage and species-specific interactions (as well as the idea that the actual interaction interfaces may evolve considerably without disrupting the basic structure of the complex).
To summarize, this study clarifies the probable role for Fip1 in the polyadenylation complex, and it reveals that this scaffold has a remarkable “structure”, one with important functional and evolutionary ramifications.
Gretchen Meinke, Chukwudi Ezeokonkwo, Paul Balbo, Walter Stafford, Claire Moore, Andrew Bohm (2008). Structure of Yeast Poly(A) Polymerase in Complex with a Peptide from Fip1, an Intrinsically Disordered Protein Biochemistry, 47 (26), 6859-6869 DOI: 10.1021/bi800204k
The RNA Underworld 