The polyadenylation complex – RNA binding proteins

As I indicated in the overview of polyadenylation, this process is mediated by a complex of proteins called, naturally enough, the polyadenylation apparatus. This machinery has been reviewed many times over the years, and the review I pointed to earlier provides a nice overview of the subunits involved. An illustration from this review is below the fold at the end of the essay; this illustration and the review will serve as the source for much of this information, and will take the place of what would be a long list of citations that pertain to the details that follow. Rather than re-invent the wheel, what I thought I would do is to summarize things taking a different approach. Thus, what I will try to do in the next few essays is to discuss things from the perspective of the biochemical activities one finds associated with the complex, with special attention being paid to unexpected or unexplained aspects of the complex. As well as being a list, I hope that these essays will raise in readers’ minds one or two questions about the process, questions likely without answers at the moment.

RNA binding proteins

As I indicated before, the site of processing and polyadenylation of a pre-mRNA is established by the coincidence of several distinctive cis elements in the RNA, collectively known as the polyadenylation signal.  As one might expect, the processing complex possesses a number of RNA-binding subunits. In mammals, these include the 160 kD and 100 kD subunits of the Cleavage and Polyadenylation Specificity Factor (abbreviated in this blog as CPSF160 and CPSF100, respectively), the 64 kD subunit of Cleavage stimulatory Factor (CstF64), the 25 kD subunit of Cleavage Factor II (CFIm25), CPSF30, and Fip1.  What is interesting about this collection is that there are only three components of the mammalian polyadenylation signal – the poly(A) signal AAUAAA, the downstream element, and the upstream sequence element.  These three components have been associated with CPSF160, CstF64, and CFIm25, respectively.  Fip1 has been shown to associate with the actual processing site itself.  CPSF100 and CPSF30 have not been “localized” to any particular part of the pre-mRNA (although the yeast counterpart of CPSF30, Yth1, has been shown to associate with the cleavage site itself, much as does the human Fip1).

A similar situation is seen in yeast.  Thus, Yhh1 (the yeast counterpart of CPSF160), Ydh1 (=CPSF100), Yth1 (=CPSF30), Rna15 (=CstF64), and Hrp1 have all been found to possess RNA-binding activity. Rna15 has been associated with the so-called positioning element, Hrp1 with the efficiency element, and Yth1 with the processing site.  Curiously, Rna15 is the ortholog of CstF64; while the latter mediates the action of the downstream element, the former binds to the yeast element that seems to be analogous to the AAUAAA polyadenylation signal.  The significance of this difference has yet to be explained.  Also of interest is the observation that the yeast Fip1 ortholog does not seem to bind RNA. 

In both mammals and yeast, there are three parts of a polyadenylation signal (four, if one counts the actual processing site).  However, the respective complexes possess more RNA binding proteins than known and functioning RNA cis elements in a polyadenylation signal. As indicated, some of these proteins have been associated with specific elements, while others have not.  The latter may be because the experiment has not been done, or instead because the tests yielded negative results that were never published.  In  any case, this “excess” of RNA-binding subunits raises some interesting questions – are there different complexes that have different subunit compositions?  Is there a remodeling of the RNA-protein interactions during the course of the reaction?  Might relatively non-specific RNA-protein interactions be important for the functioning of these proteins?

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This is the figure I promised above the fold – it is from the review by Proudfoot and O’Sullivan

Figure 1 -taken from Proudfoot and O’Sullivan. Comparison of cleavage-polyadenylation factors associated with poly(A) signals in mammals and budding yeast.

In mammals (A), the five factors involved are indicated, showing the subunit structures and molecular weights of CPSF and CstF. The polymerase II CTD (grey) is also shown. In yeast (B), three multi-subunit factors are involved with the subunit components indicated. The positions of the different factors are intended to indicate (where known) the interactions between different factors and their subunits. The sequence elements that comprise the poly(A) signals are also indicated by black rectangles, and the site of cleavage (and subsequent polyadenylation) is shown by a red arrow bolt. Homologous factors between yeast and mammals are color matched. Known homologous subunits between yeast and human include: Pta1p=symplekin; Pfs2p=CstF₅₀; Ysh1p=CPSF₇₃; Ydh1p=CPSF₁₀₀; Yth1p=CPSF₃₀; Yhh1p=CPSF₁₆₀; Pap1p=PAP; Rna14p=CstF₇₇; Rna15p=CstF₆₄[3]. Clp1p and Pcf11 are components of CFIIm [4].

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Proudfoot and O’Sullivan, Curr. Biol. 12, R855-R857, 2002.

references 3 and 4 from Proudfoot and O’Sullivan (cited in the legend to the figure):

3 Shatkin, A.J. and Manley, J.L. (2000). The ends of the affair: Capping and polyadenylation. Nat. Struct. Biol. 7, 838-842. [Medline]

4 de Vries, H.,Ruegsegger, U.,Hubner, W.,Friedlein, A.,Langen, H. and Keller, W. (2000). Human pre-mRNA cleavage factor IIm contains homologs of yeast proteins and bridges two other cleavage factors. EMBO J. 19, 5895-5904. [Medline]