Poly(A) polymerase and poly(U) polymerase tailing bias against 2′-O-methylated small RNA 3′-ends. (A) Gel analysis of small RNA polyadenylation. Products of poly(A) polymerase (PAP), or poly(U) polymerase (PUP) polyadenylation reactions were resolved by denaturing PAGE and visualized by SYBR Gold staining. Tailing reactions contained 21-nt RNAs that were unmodified (2′-OH) or 2′-O-methylated (2′-O-Me) as indicated. (B) Quantification of 2′-O-methyl 3′-end nucleotide bias. Small RNA polyadenylation reactions using PAP or PUP were performed on unmodified or 2′-O-methylated small RNAs that terminated with A, C, G, or U. The extent of polyadenylation was determined by densitometry. Plotted points represent the mean ± standard error of the mean (SEM); n = 3 experimental replicates.
