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

Structural requirements for the cleavage activity. (A,B) The HCC lacking CstF64 (HCC³⁵³-64) was analyzed by SDS/polyacrylamide gel electrophoresis followed by Coomassie blue staining (panel A) and tested for the ability to support processing in a reaction additionally containing NTD, WT core U7 snRNP, FLASH, and SLBP (panel B). The input mH2a* pre-mRNA and processing in the absence of the NTD are shown in lanes 1,2 of panel B, respectively. (C,D) As in panels A and B, with the exception that symplekin in HCC⁵³⁸-64 begins with amino acid 538. (E) Processing of mH2a* pre-mRNA in the presence of SLBP and recombinant U7 snRNP containing the NTD, WT core U7 snRNP, FLASH, and HCC³⁵³. In lanes 4–6, mPSF was added at two-, four-, and eightfold molar excess relative to the HCC³⁵³. (F,G) As in panels C and D, with the exception that CPSF100 in HCC^538,ΔPIM-64 lacks amino acids 460–486 interacting with CPSF160 and WDR33. (H,I) Residues of the NTD that make contact with CPSF100 (panel H) were replaced with alanines in two overlapping blocks and the resultant mutant NTDs were tested for the ability to support processing of mH2a* pre-mRNA together with WT core U7 snRNP, FLASH, HCC³⁵³, and SLBP (panel I). (J) As in panel I, with the exception that the mutated residues, Leu45 and Met116, were located at the interface of the NTD interacting with SmE, and each mutant NTD was added to the processing reaction to reach 5× and 1× molar ratio relative to the HCC³⁵³.