The polyadenylation complex – Endonucleases

Messenger RNA 3’ end formation involves an RNA processing step.  It is thus natural to assume that the polyadenylation complex (as illustrated here) includes at least one subunit that is an endonuclease (an enzyme that cuts the RNA within the polynucleotide chain, as opposed to at the 5′ or 3′ ends).  Indeed, the complex does include such a nuclease.  However, just as there seem to be more RNA binding proteins than known cis element, there may be a multiplicity of endonucleases in the complex.  Specifically, at least two CPSF subunits (CPSF73 and CPSF30) have been shown to possess endonucleolytic activity in vitro (1-4).  The research community would probably consider that CPSF73 is the most probable candidate for THE processing endonuclease, but the possibility that CPSF30 serves this role, along with or instead of CPSF73, cannot be ruled out.  (Except perhaps in yeast, whose CPSF30 counterpart, Yth1, apparently lacks the nuclease activity seen in other eukaryotic homologs [5].)

The nuclease situation may be more complicated than this.  CPSF73 and CPSF100 are themselves related, and are members of the metallo-beta-lactamase family of nucleases.  CPSF100 lacks some amino acid side chains that have been implicated in nuclease activity in CPSF73 (1,6), suggesting that this subunit is probably not the processing endonuclease.  However, bacterial nucleases of the metallo-beta-lactamase family, so-called Rnase J enzymes, have been found to be both endo- and exo-nucleases (7); the latter activities are 5’->3’ in their directionality.  To my knowledge, a detailed comparative analysis of Rnase J’s and CPSF100/73 that accounts for these distinct activities has not been performed, but the possibilities as far as the existence of even greater numbers of nuclease activities in CPSF are tantalizing.  Indeed, the suggestion made by Wickens and Gonzalez some years ago ( 8 ), that RNA processing and 3’ end formation in the nucleus may involve a versatile Swiss army knife of nucleases, seems as relevant as ever today.

Readers may wonder – how specific are these nucleases?  By themselves, not very.  But in the cell, they have a rather exquisite specificity.  This probably reflects the fact that these enzymes are almost certainly complexes with other proteins that serve to “bring” the nuclease to its RNA substrate.  But another possible parallel with bacteria may also be in play with these nucleases.  It turns out that bacteria have collections of nucleases that exist in concert with anti-nucleases (9) – specific partners that inhibit (and thus regulate) the activity of the nuclease.  A similar situation is seen with the Arabidopsis CPSF30 and Fip1 orthologs (4).

To close this brief summary, two points may be made.  One is that, as is the case with RNA binding proteins (and other members of the complex that will be discussed at some time in the future), there may be a multiplicity of nucleases within the polyadenylation complex.  The second is that the interesting sequence similarities and functional parallels with bacterial nucleases may be a first halting glimpse into the origins and evolution of the polyadenylation complex.  This too will be a subject for future essays.

References:

1.  Ryan K, Calvo O, Manley JL. (2004) Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease. RNA 10, 565–573.

2.  Mandel CR, Kaneko S, Zhang H, Gebauer D, Vethantham V, Manley JL, Tong L. (2006) Polyadenylation factor CPSF-73 is the pre-mRNA 3′-end-processing endonuclease. Nature 444, 953–956.

3. Bai C, Tolias PP. (1996) Cleavage of RNA hairpins mediated by a developmentally regulated CCCH zinc finger protein. Mol Cell Biol 16, 6661–6667.

4. Addepalli B, Hunt AG. (2007) A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor. Nucleic Acids Res 35, 4453-4463.

5.  Addepalli B, Hunt AG. (2008 ) Ribonuclease activity is a common property of Arabidopsis CCCH-containing zinc finger proteins. FEBS Lett 582, 2577-82.

6.  Callebaut I, Moshous D, Mornon JP, de Villartay JP. (2002) Metallo-ß-lactamase fold within nucleic acids processing enzymes: The ß-CASP family. Nucleic Acids Res 30, 3592–3601.

7.  de la Sierra-Gallay IL, Zig L, Jamalli A, Putzer H. (2008 ) Structural insights into the dual activity of RNase J. Nat Struct Biol 15, 206-212.

8.  Wickens M, Gonzalez TN. (2004) Molecular biology. Knives, accomplices, and RNA. Science 306, 1299–1300.

9.  Condon C. (2007) Maturation and degradation of RNA in bacteria. Curr Opin Microbiol 10, 271-8.