Nucleotide-adding activity of S. pombe Cid1 and S. cerevisiae Poly(A) polymerase (PAP) on various model RNA substrates. RNA was incubated with enzyme and rNTPs as indicated and run on denaturing polyacrylamide gels, stained with SYBR Gold and imaged on a Typhoon scanner. Markers are DNA with the indicated chain lengths for the equivalently migrating RNA, using the ∼1.04× greater mass to charge ratio of RNA per residue. For example, a 500 nt DNA marks the approximate migration of a 482 residue RNA. (A) Cid1 adds long tails of A, U, or I, but much shorter tails of C or G to A24. (B) PAP adds long tails of A, a 35–50 nt stretch of G, but only short stretches of C, U, or I to A24. (C) Cid1 efficiently adds long stretches of A or U, but mostly just 50 residues of I to a model poly(A)+ mRNA, whereas it inefficiently adds A, U, or I to a nonpolyadenylated RNA. (D) PAP efficiently adds A to either polyadenylated or nonpolyadenylated RNA (lanes 3 and 6) and adds a short stretch of I to poly(A)+ RNA, but only inefficiently adds U or I to nonpolyadenylated RNA.
