Figure 3.

Characterization of the 3′ nucleotidyl phosphatase activity of 20S editosomes. (A) A 31 nt ssRNA molecule (RNA31) was radioactively labeled (*) at its 3′ end using 5′-[³²P]-pCp and incubated for up to 3 h with 20S editosomes or alkaline phosphatase (AP). Reaction products were resolved in urea containing polyacrylamide gels and are depicted in the margin to the right. (B) Plot of the dephosphorylation (dfos) activity of 20S editosomes at different pH values. (C) Dephosphorylation of RNA-editing substrate molecules as part of an RNA-editing reaction cycle. Left panel: Precleaved insertion editing of 20S editosomes using a 3′ phosphorylated 5′ pre-mRNA cleavage fragment (5′ CF). Mock, mock control; T4 RNA ligase, control ligation with T4 RNA ligase. Right panel: Precleaved insertion editing of 20S editosomes using a 3′ dephosphorylated 5′ CF. Reaction products and intermediates are sketched in between the two gels. The mock sample contains a single band and degradation was measured <1%.