Figure 1.
Ccr4-Not subunits associate with the arginine methyltransferase Hmt1.
(A) Cells expressing the indicated Myc-tagged Ccr4-Not subunit as well as HA-tagged Hmt1 were used to examine the interaction between the Ccr4-Not complex and Hmt1. Cells were grown and lysates prepared as described in Materials and Methods. Ccr4-Not subunits were immunoprecipitated with α-Myc antibody (α-Myc IP) and the co-immunoprecipitation of Hmt1 was assessed by immunoblotting with an α-HA antibody (α-HA). The efficiency of immunoprecipitation was assessed by probing the same blots with α-Myc antibody (α-Myc). Input samples (30 µg total lysate) were probed with α-HA antibody to detect HA-tagged Hmt1 (α-HA). As controls, cells expressing no tagged protein (Control) or only Hmt1-HA show no co-immunoprecipitation. (B) The same samples in (A) were subjected to immunoprecipitation with α-HA antibody (α-HA IP) to detect co-immunoprecipitation with the Myc-tagged Ccr4-Not subunits. Immunoblots were probed with α-Myc antibody (α-Myc) to detect Ccr4-Not subunits or α-HA antibody (α-HA) to assess the level of Hmt1 precipitated. Input samples (30 µg total lysate) were probed with α-Myc to detect Ccr4-Not subunits (α-Myc). As controls, cells expressing no tagged protein (Control) or only Hmt1-HA show no co-immunoprecipitation.
Figure 2.
Association of the hnRNP proteins Hrp1 and Nab2 with Ccr4-Not subunits depends on Hmt1 arginine methyltransferase activity.
(A) Nab2 associates with Caf1, Ccr4, and Not5. Cells expressing the indicated Myc-tagged Ccr4-Not subunit were used in co-immunoprecipitation experiments as described in the Methods to examine the interaction between the Ccr4-Not complex and Nab2. As a control, cells expressing no tagged protein (Control) show no co-immunoprecipitation. Nab2 co-immunoprecipitation and input levels (30 µg) were detected by α-Nab2 immunoblotting while Ccr4-Not subunits detected by α-Myc immunoblotting. (B) Hrp1 associates with Caf1, Ccr4, Not1, Not2 and Not5. Experiments were performed as in (A) and the co-immunoprecipitation of Hrp1 was assessed by immunoblotting with α-Hrp1 antibody (α- Hrp1). C, The interaction between Hrp1 and Caf1 is not RNase-sensitive. Cells expressing Caf1-Myc were grown and lysed as described in Materials and Methods. Lysate was divided into two samples and one aliquot was treated with RNase A prior to immunoprecipitation with α-Myc antibody as described in the Methods. Fold change in co-immunoprecipitation was determined using reverse image scanning densitometry as described in the Methods. D, Caf1 association with Hrp1 depends on Hmt1-mediated arginine methylation. CAF1-MYC hmt1Δ cells were transformed with empty vector, HMT1 or hmt1G68R expression vectors, and cells were grown in SC-Ura media to select for plasmid maintenance. Lysates were prepared as described in Materials and Methods. Caf1-Myc was immunoprecipitated with α-Myc antibody (α-Myc IP) and the co-immunoprecipitation of Hrp1 was assessed by immunoblotting with α-Hrp1 antibody (α-Hrp1). Efficiency of immunoprecipitation was assessed by probing the same blots with α-Myc antibody. Input samples (30 µg total lysate) were probed with α-Myc and α-Hrp1 antibody. Fold change in co-immunoprecipitation was determined using reverse image scanning densitometry as described in Methods.
Table 1.
Ccr4-Not TAP purifications identify Mlp1/2 and Sam1/2.
Figure 3.
Not1 co-associates with the nuclear basket subunit Mlp1.
(A) Whole-cell extracts from cells individually expressing NOT1-Myc or MLP1-HA or from cells expressing both tagged alleles were used in α-HA immunoprecipitation experiments to pull-down Mlp1. Co-associated Not1 was detected by α-Myc immunoblot which was subsequently stripped and reprobed with α-HA to detect immunoprecipitated Mlp1. (B) Input samples (30 µg) were initially probed with α-Myc to detect Not1 levels and then the membrane was stripped and reprobed with α-HA to detect Mlp1.
Table 2.
Ccr4-Not TAP purifications identify Mlp2 and mRNA processing and export factors.
Figure 4.
Not4 and not4L35A overexpression cause differential growth effects in NPC mutant cells.
(A) Wild-type and nup116Δ cells were transformed with empty vector, CCR4 or NOT4 overexpression constructs. Cells were grown to saturation in SC-Ura media, ten-fold serially diluted, and spotted onto SC-Ura plates. Plates were incubated at 25°C or 30°C. (B) Whole cell extracts of cells transformed with empty vector, CCR4, NOT4, or not4L35A were analyzed by immunoblotting and probed with α-FLAG antibody. Blots were probed with α-PGK1 antibody as a loading control. (C) Wildtype, nup1Δ, nup116Δ, nup120-1, nup133-1, and nup49-313 cells were transformed with empty vector, NOT4, or not4L35A overexpression constructs. Cells were grown to saturation in SC-Ura media, ten-fold serially diluted, and spotted onto SC-Ura plates. Plates were incubated 25°C or 35°C.
Figure 5.
Not4 and not4L35A overexpression cause differential growth effects in hnRNP mutant cells.
Wildtype, Nab2-C437S, Hrp1-P531A and npl3-1 cells were transformed with empty vector, NOT4 or not4L35A overexpression constructs. Cells were grown to saturation in SC-Ura media, ten-fold serially diluted, and spotted onto SC-Ura plates. Plates were incubated at 25°C for the permissive temperature or 39°C (for Nab2-C437S) 37°C (for Hrp1-P531A), or 30°C (for npl3-1) for the elevated temperature.
Figure 6.
Not4 overexpression exacerbates the poly(A) RNA export defect in nup116Δ cells.
Wildtype and nup116Δ cells were transformed with empty vector or a NOT4 overexpression construct. Cells were grown to log phase at 30°C and subjected to FISH. (A) FISH was performed on cells as described in Materials and Methods. Panels are shown for poly(A) RNA and DAPI to visualize chromatin. (B) Quantification of cells showing nuclear accumulation of poly(A) RNA. Images were analyzed blind, and a minimum of 50 cells were analyzed in triplicate for each condition. Student's t-test was used to determine statistical significance. (C) Wildtype and nup116Δ cells were transformed with empty vector or a NOT4 overexpression construct and an NLS-NES-GFP construct. Cells were grown to log phase and analyzed by live cell microscopy for GFP localization.
Table 3.
Strains and plasmids used in this study.