It’s getting crowded

One of the interesting things about the polyadenylation complex is the multiplicity of RNA-binding proteins in the system.  As I mentioned before, there are many such proteins, more than the numbers of cis elements typically associated with polyadenylation.  Closer inspection of the complex reveals more paradoxes involved RNA-binding proteins and polyadenylation.

Recently, a rotation student in my lab published a study that reported the discovery of a new RNA-binding domain in the plant polyadenylation complex (1).  There are two findings that are described this study.  The obvious one, that can be extracted from the title, is that the 77 kD subunit of the Arabidopsis Cleavage Stimulatory Complex (CstF77) is in fact an RNA binding protein.  In this report, the new RNA-binding domain was localized to the C-terminus of the protein.  This is interesting because this part of CstF77 is not conserved in its eukaryotic (yeast and mammalian) counterparts.  This raises the possibility that RNA binding by CstF77 is a plant-specific phenomenon.

The second interesting finding is the location of the RNA-binding domain. The structures of two eukaryotic CstF77 proteins have been solved, and  it has been established that the protein exists as a dimer (2,3).  The dimer is “held together” by extensive interactions involving the “middle” of the protein, and several of the characteristic “HAT” motifs (4) in the protein.  The C-termini of each subunit of the dimer probably extends into the cavity formed by the dimer, as suggested in the figure below.

Structures of the CstF77 monomer (left) and dimer (right), adapted from reference 1. On the left, the N-terminus of the protein is colored yellow and the middle part purple. On the right, just the interweaving of the two middle parts is shown. The places where the C-termini will protrude are shown with arrows; the C-termini are not included in the solved structures.

It turns out that the RNA-binding domain of CstF77 is  not the only RNA-binding activity associated with the C-terminus of the protein.  The C-terminal region is also involved for the interaction between CstF77 and CstF64 in mammals and plants.  Also in plants, Fip1 (another RNA-binding protein) binds to the very C-terminus of CstF77 (5).  As is shown in the paper that is the focus of this essay, CPSF30 also binds to the C-terminus of CstF77 in plants.  Taking into account the dimeric nature of CstF77, this places as many as eight RNA-binding sites within the CstF77 cavity.  This is many more sites than sub-elements within the plant polyadenylation signal.

Many questions arise from these considerations.  Are all of these RNA-binding sites present in each and every individual polyadenylation complex?  Or are many of these sites mutually exclusive (it is, after all, hard to imagine so many proteins being squeezed into that small space)?  What are the RNA-binding properties of the combined collection(s) of binding sites?  Is it possible for the complex to remodel itself in the course of the reaction, exchanging RNA-binding sites along the way? Of course, I don’t have answers to these questions.  Which is not a bad thing, since the questions will keep me busy for many years to come.

References:

1.  Bell, S. A., and Hunt, A. G..  The Arabidopsis ortholog of the 77 kDa subunit of the cleavage stimulatory factor (AtCstF-77) involved in mRNA polyadenylation is an RNA-binding protein.  FEBS Lett. 584, 1449-54.

2.  Legrand, P., Pinaud, N., Minvielle-Sebastia, L. and Fribourg, S. (2007). The structure of the CstF-77 homodimer provides insights into CstF assembly. Nucleic Acids Res 35, 4515-22.

3.  Bai, Y., Auperin, T.C., Chou, C.Y., Chang, G.G., Manley, J.L. and Tong, L. (2007). Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors. Mol Cell 25, 863-75.

4.  Preker, P.J. and Keller, W. (1998). The HAT helix, a repetitive motif implicated in RNA processing. Trends Biochem Sci 23, 15-6.

5.  Addepalli, B. and Hunt, A.G. (2008). The interaction between two Arabidopsis polyadenylation factor subunits involves an evolutionarily-conserved motif and has implications for the assembly and function of the polyadenylation complex. Protein Pept Lett 15, 76-88.