Studies with recombinant U7 snRNP demonstrate that CPSF73 is both an endonuclease and a 5′–3′ exonuclease

The release of the 5′ terminal nucleotide during degradation of the DCP RNA by recombinant U7 snRNP. (A) Nucleotide sequence of the DCP RNA substrate used in the assay. The asterisk indicates the position of ³²P label and the HDE is underlined. (B,C) The release of the 5′ terminal nucleotide during incubation of the DCP RNA with indicated components. The input DCP RNA is shown in lanes 1,10 of panel B and lane 1 of panel C. (D) The effect of SSU72 on the release of the 5′ terminal nucleotide during degradation of the DCP RNA in the presence of recombinant U7 snRNP consisting of HCC³⁵³, WT core U7 snRNP, FLASH, and either wild-type (WT, lanes 3–7) or R185A mutant NTD (lanes 8–12). The numbers indicate concentration of SSU72 in µM. The input DCP RNA and the degradation in the absence of the NTD are shown in lanes 1 and 2, respectively. (E) The release of the 5′ terminal nucleotide during incubation of the DCP RNA with mouse nuclear extract (NE) either alone or in the presence of increasing amounts of SSU72 (0.75–20 µM range, as indicated).