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. 2018 Sep 26;46(22):12022–12039. doi: 10.1093/nar/gky862

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — CstF-77 and CstF-50 enhance MCP-CstF-64-dependent cleavage and polyadenylation. (A) Schematic representation of CstF-77-Myc (CstF-77 with three Myc tags at its carboxy-terminal end) and HA-CstF-50 (CstF-50 with three hemagglutinin tags at its amino-terminal end). The position of the nuclear localization sequence (NLS), position and the number of the carboxy- and amino-terminal HATs, MT and the CTD of CstF-77 are shown. Similarly, the seven WD40 (WD40) repeats and the dimerization (Dim) domains are shown for CstF-50. (B) Cartoon of the interactions between MCP-CstF-64, CstF-77, CstF-50 and SL-Luc reporter construct. (C) Increased amounts of CstF-77-Myc increase C/P as measured by SLAP. Western blots with an antibody against Myc (WB: Myc), against CstF-77 (WB: CSTF3), against MCP-CstF-64 (WB: FLAG) or CstF-64 (WB: CSTF2) are shown. Antibodies against β-tubulin were used as a loading control. (D) Decreased CstF-77 reduces C/P as measured by SLAP. Hela cells were transfected with either a non-specific (scr) or CSTF3-specific siRNAs. Twelve hours after siRNA transfection, cells were transfected for SLAP. Western blots show the reduced expression of the endogenous CstF-77, MCP-CstF-64 (WB: FLAG). (E) HA-CstF-50 co-expressed with MCP-CstF-64 and CstF-77-Myc increases C/P as measured by SLAP. Western blots with the respective antibodies to verify expression of the exogenous proteins through their tags (WB: FLAG, Myc, HA) and antibodies to show relative expression to the endogenous proteins (WB: CSTF1, CSTF2, CSTF3). (F) Mutations in CstF-77-Myc that disrupt the interaction of CstF-77 with CstF-50 reduce C/P as measured by SLAP. Two-point mutations were made in CstF-77 (P584A, M589A, collectively called +dm77) that disrupt the interaction of CstF-77 with CstF-50. Immunoprecipitation with an antibody against Myc tag: no CstF-77, CstF-77-Myc (+77) and CstF-77-Myc-dm77 (+dm77). Western blots for Myc tag (WB: Myc), and endogenous CstF-50 (WB: CSTF1) and β-tubulin were presented. (G) Mutations in MCP-CstF-64 that disrupt the interaction of CstF-64 with CstF-77 decrease both SLAP and the stimulatory effect of CstF-77. MCP-CstF-64-ΔHinge lacks amino acids 96–216 from CstF-64. MCP-CstF-64 (R159P) is a point mutation in the Hinge domain that interrupts the interaction with CstF-77, MCP-CstF-64 (H4) is a series of point mutations in the fourth α-helix of the Hinge domain that interrupt interaction with CstF-77.