Figure 6. MBLAC1 shows specific endoribonucleolytic activity on histone pre-mRNA recognising specific sequence/structural RNA elements in vitro.

(A, C–F) Time-dependent in vitro cleavage assays using [γ-³²P] ATP 5' labeled histone 2H3C RNA fragments (RNA 1–6) in the presence of HEK293 produced wtMBLAC1. (B) Time-dependent in vitro cleavage assays using [γ-³²P] ATP 5' labeled histone 2H3C RNA 2 (50 nt) in the presence of HEK293 produced wild-type or mutMBLAC1. Cleavage products of RNA 2 after incubation with wtMBLAC1 (WT) are compared with those after incubation with mutMBLAC1 (MUT). Cleavage products of the RNA 1 are compared with those of the differently modified fragments. Schematic views of the RNA fragments are shown. The schematics show the numbering assigned to the possible cleavage sites present in each RNA; arrows indicate the physiologically major (red) and minor (blue) cleavage sites. The CA dinucleotide corresponding to the major cleavage site is in red. Sequence or structural modifications of each RNA are in yellow. The size of processed RNA products is indicated when appropriate. Unprocessed RNA fragments corresponding to nucleotides 1 to 29 and 1 to 27 of the WT RNA 1 were used as mass markers and indicated as 29 and 27, respectively. Note that the 29 and 27 markers migrate more slowly on gel electrophoresis compared to the corresponding fragments generated by MBLAC1 catalysis in positions 27 and 29. This observation is explained by the presence of an extra negatively charged phosphate group on the 5'end of the products generated by MBLAC1 catalysis, compared to the markers, as expected during maturation of histone transcripts (Furger et al., 1998).