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 mportant 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.
Monarez
Roberto R
RR
Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
MacDonald
Clinton C
CC
Dass
Brinda
B
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D039241:Cleavage Stimulation Factor / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D026723:Polyadenylation; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
D001704:Biopolymers; D039241:Cleavage Stimulation Factor; C492658:Cstf2t protein, mouse; D012313:RNA