Waghray S , Williams C , Coon JJ , Wickens M .

GM31892 NIGMS NIH HHS , GM50942 NIGMS NIH HHS , GM80148 NIGMS NIH HHS , R01 GM050942 NIGMS NIH HHS , R37 GM031892 NIGMS NIH HHS , R01 GM080148 NIGMS NIH HHS , R01 GM031892 NIGMS NIH HHS

	FIGURE 1. Mutational analysis of CAF1 proteins reveals requirement for NOT1 interaction. (A) Residues mutated are depicted on the human CAF1a structure (PDB 2D5R) and colored by the interaction disrupted. (B) Outline of the tethered function assay. (FF) firefly luciferase, (Ren) Renilla luciferase. (C) Results of tethered function assay, normalized to “no MS2 protein” control (none). Three independent experiments were performed, with four oocyte replicates each time. Error bars represent 1 SD. Student's two-tailed t-test was used to determine significant changes between CAF1 WT and mutants, and only NOT1-interaction-defective mutants were significantly (P < 0.005) different. (D) Outline of coimmunoprecipitation assay from Xenopus oocytes. (E) NOT1 interacts with wild-type CAF1 but not with the M141R mutant. A CAF1-interaction-defective mutant of NOT1 served as a negative control. Western blots depict a single representative experiment, and three biological replicates were conducted.
	FIGURE 2. NOT1 mediates interactions between CAF1 and its protein partners. (A) Schematic representation of GST pull-down assay. (B) GST-CAF1 proteins are efficiently pulled down. Western blots with antibodies to known regulatory proteins were performed after GST pull-down to test for CAF1 interaction. Western blots depict a single representative experiment, and three biological replicates were conducted. (C) Graphical representation of mass spectrometry data from three biological replicates for GST-CAF1a wild type and mutant. P-values calculated using a two-tailed Student's t-test are indicated by dot size. Members of the CCR4–NOT complex (boxed) interact strongly with CAF1a WT but not the M141R mutant. Xp54 is also enriched specifically for CAF1a WT over the M141R mutant, but to a lesser extent. (D) Same as in C, but with CAF1b.
	FIGURE 3. NOT1 MIF4G is required for CAF1 interactions in vivo. (A) Schematic of the coimmunoprecipitation assay with HA-CAF1 as the bait protein. (B) Western blots with antibodies to proteins that interact with CAF1 by GST pull-down were conducted to test interaction by co-IP. CCR4 is a positive control, and eIF4E is a negative control. Western blots depict a single representative experiment, and three biological replicates were conducted. (C) Schematic of the coimmunoprecipitation assay with HA-MIF4G (the NOT1 MIF4G domain) as the bait protein. Both HA-MIF4G WT and a CAF1-binding deficient mutant of the MIF4G domain (HA-MIF4G mut) were used. myc-CAF1 was coexpressed as a control. (D) Western blots were conducted to test whether 4E-T and Xp54 interact with the NOT1 MIF4G domain. eIF4E was used as a negative control, and myc-CAF1 also served as a control. Western blots depict a single representative experiment, and three biological replicates were conducted.
	FIGURE 4. NOT1 MIF4G-Xp54 interaction is required for repression. (A) Schematic of tethered function assay with NOT1 fragments. (FF) firefly luciferase, (Ren) Renilla luciferase. (B) NOT1 fragments used in the tethered function assay. Mutations were made in the NOT1 MIF4G domain to obtain Xp54- and CAF1-binding defective constructs, as indicated by asterisks. Full-length NOT1 is depicted for scale. (C) Results of tethered function assay, normalized to “no MS2 protein” control (none). Xp54 was used as a control for repression. Three independent experiments were performed, with four oocyte replicates each time. Error bars represent 1 SD. Student's two-tailed t-test was used to compare the NOT1 amino terminus to amino terminus (−) and the wild-type MIF4G to the Xp54-binding defective MIF4G. Both were significantly (P < 0.005) different. (D) Schematic of the coimmunoprecipitation assay with HA-MIF4G (the NOT1 MIF4G domain) as the bait protein. HA-MIF4G WT, the CAF1-binding deficient mutant of the MIF4G domain, and the Xp54-binding deficient mutant were used. (E) Western blots were conducted to test whether 4E-T and Xp54 interact with the Xp54-binding deficient MIF4G domain. Western blots depict a single representative experiment, and three biological replicates were conducted. (F) Model of the interactions between NOT1, CAF1, Xp54, and 4E-T.
	FIGURE 5. 4E-T mediates repression in an eIF4E-independent manner. (A) 4E-T protein depicted to show eIF4E-binding motif (black box) and mutants used for modified tethered function assay. Table is used to show the different repression mechanisms of 4E-T (“Mechanism 1,” eIF4E-dependent and “Mechanism 2,” eIF4E-independent). (B) Schematic of modified tethered function assay with coexpressed 4E-T proteins. (FF) firefly luciferase, (Ren) Renilla luciferase. 4E-T represents the wild-type and mutant 4E-T constructs expressed, with 1 and 2 indicating the repression mechanisms from panel A. “Protein” represents CAF1b, Xp54, or NOT1 MIF4G. (C) Results of tethered function assay, normalized to “no MS2 protein” control (none). Black bars indicate no coexpressed 4E-T protein, gray shaded bars indicate wild-type 4E-T protein, the eIF4E-interaction-defective allele of 4E-T (YLL-AAA), and the 4E-T 1–180 truncation. Three independent experiments were performed, with four oocyte replicates each time. Error bars represent 1 SD. Student's two-tailed t-test was used to compare the 4E-T 1–180 data to the 4E-T WT data. For CAF1b and Xp54, the difference was highly significant (P < 0.005), while the NOT1 MIF4G domain had a slightly less significant difference of P < 0.05. (D) Graph of Renilla luciferase translation from the same experiment to show effects of the 4E-T coexpressed proteins on general translation. (E) Schematics depicting the summary of the tethered function assay data. “Protein” represents CAF1b, Xp54, and NOT1 MIF4G.
	FIGURE 6. Xp54 and CAF1 repress translation independent of eIF4E and the 5′ cap. (A) Schematic of the tethered function assay. (FF) firefly luciferase, (Ren) Renilla luciferase. “Protein” represents GLD2-D242A (negative control), CAF1b, CAF1b DE-AA, or Xp54. (B) Schematic of the firefly luciferase reporters used. The IRES translation factor requirements are depicted. (C) Results of tethered function assay, normalized to “no MS2 protein” control (none, black bars). Three independent experiments were performed, with four oocyte replicates each time. Error bars represent 1 SD. Student's two-tailed t-test was used to compare the Xp54, CAF1b, and CAF1b DE-AA data to the no MS2 protein data for each reporter. For CAF1b, CAF1b DE-AA, and Xp54, the difference was highly significant (P < 0.005) only for the capped and PV IRES reporters.
	FIGURE 7. The NOT1 MIF4G domain recruits Xp54 and 4E-T to mediate eIF4E- and cap-independent repression by CAF1. (A) Model of repression when a tethered repressor is on the 3′ end of a reporter mRNA. (B) We propose that repression may occur in a similar manner by RNA-binding proteins that recruit the CAF1/NOT1/Xp54/4E-T complex when bound to target mRNAs.

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