Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers.
CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, tauCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between tauCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of tauCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and tauCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)(18), poly(A)(18) or poly(C)(18), with affinity for poly(C)(18) being the lowest. However, CstF-64 had a higher affinity for poly(U)(18) than tauCstF-64, whereas it had a lower affinity for poly(GU)(9). Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)(18), but changes in amino acids downstream of the C-terminal alpha-helical region decreased affinity towards poly(U)(18). Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.[1]References
- Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers. Monarez, R.R., Macdonald, C.C., Dass, B. Biochem. J. (2007) [Pubmed]
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