plasmid pcdna3 n flag masc1  (Addgene inc)

Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: a The bulk of Hel2-RM binds to disomes and trisomes Polysome profiles with extracts derived from Hel2-RM g-log (orange) or Hel2-RM g-log/RNase (magenta). b Hel2-RM binds to ribosomes in a salt-resistant manner. Ribosome binding assays with extract of wild type log or Hel2-RM log as described in Fig. . Shown is the mean of 3 independent experiments (bars) and the result of each experiment (dots) with the standard deviation. An example blot is shown in Fig. . c High affinity binding of Hel2 to disomes depends on Asc1. Polysome profiles with extracts of ∆ asc1 log (orange) or ∆ asc1 log/RNase (magenta). d Hel2 binds to ∆ asc1- disomes in a salt-sensitive manner, with or without induction of collision. Ribosome-binding assays were performed with extract of ∆ asc1 log (no-collision, blue) or ∆ asc1 coll (collision, light blue). Shown is the mean of at least 3 independent experiments (bars) and the result of each experiment (dots). An example blot is shown in Fig. . Polysome profiles shown in a and c , including wild type log/RNase A 260 traces (dotted gray lines) and Hel2 distribution profiles of wild type log/RNase (gray), were recorded side by side. Labeling is as detailed in Fig. .

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Derivative Assay, Binding Assay, Standard Deviation, Labeling

Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: Polysome profiles and Hel2 distribution profiles of RNase-treated extracts were recorded side by side (see Methods). To avoid overloading of immunoblots, loading of fraction 2 was reduced to 50%. Peak deconvolution and quantification of monosomes, disomes, and trisomes is described in Methods. Estimated numbers of disomes and trisomes per cell are based on the assumption that a yeast cell harbors approximately 300,000 ribosomes . Shown are A 260 profiles (colored solid lines), Hel2 immunoblots, Hel2 distribution profiles (dotted black lines), and the distribution of monosomes, disomes, and trisomes obtained by peak deconvolution (colored areas under the A 260 profiles) of ( a ) wild type log/RNase ( b ) wild type coll/RNase ( c ) ∆ asc1 log/RNase ( d ) Hel2-RM g-log/RNase . e Hel2 occupancy on translating monosomes, disomes, and trisomes. The percentage of total Hel2 (approximately 3000 molecules/cell ) associated with monosomes (fractions 7–9), disomes (fractions 10–12), and trisomes (fractions 13 and 14) was calculated as described in Methods and Supplementary Data .

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Western Blot

Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: a Steady state expression of Hel2 is reduced in ∆ asc1 cells. The Hel2 expression level in wild type or ∆ asc1 lysate was analyzed by immunoblotting with α-Hel2. An example blot is shown in Fig. . For each replicate the intensity of the Hel2 band in the wild type lysate was set to 100%. Shown is the mean of 5 experiments and the result of each experiment (dots) with the standard deviation and the result of a paired samples t-test. b Turnover of Hel2 in wild type and ∆ asc1 cells. After addition of high-dose CHX aliquots were withdrawn from cell cultures and were analyzed by immunoblotting with ∝-Hel2 and α-Ubp6 (loading control). For details see Methods. c Exponential decay fit of CHX-chase repeats as shown in b . Shown is the mean of four independent experiments. Band intensities were normalized to time point 0 of wild type or ∆ asc1 , respectively. The average half life (t½) of Hel2 in wild type (gray) and ∆ asc1 (light blue) is indicated. d Inhibition of the proteasome with MG132 stabilizes Hel2. CHX-chase experiments were performed with wild type* (∆ erg6 ) and ∆ asc1* (∆ asc1 ∆ erg6 ) strains mock-treated (ctrl) or pre-treated with MG132 (MG) as described in Methods. At the time points indicated, aliquots were withdrawn and were analyzed by immunoblotting with α-Hel2 and α-Stm1 (loading control). e Exponential decay fit of four independent CHX chase experiments as shown in d . Band intensities were normalized to the time point 0 of each replicate. The average half life (t½) of Hel2 in wild type* (beige) and ∆ asc1* (dark blue) is indicated. Samples pre-treated with MG132 (MG) are labeled in red.

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Expressing, Western Blot, Standard Deviation, Control, Inhibition, Labeling

Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: a A fraction of Rps20 is polyubiquitinated in unstressed log-phase cells. Lysate from wild type or S20-H 6 cells carrying single K6R (RK), K8R (KR), or double K6R, K8R (RR) mutations. Immunoblots were analyzed with α-Rps20 (left panel, Fig. ) or α-HIS (right panel). The length of ubiquitin chains (ubi 1– 4 ) attached to S20-H 6 is indicated. Background bands above 40 kDa on α-HIS immunoblots are labeled with gray asterisks. b CHX induces polyubiquitination of S20-H 6 in a concentration dependent manner. Log-phase S20-H 6 cells were treated for 10 min with the indicated CHX concentration. c CHX-induced polyubiquitination of S20-H 6 reaches a plateau. S20-H 6 cells were treated with low-dose CHX (collision) or were left untreated (no-collision). d Polyubiquitination of Rps20 in log-phase cells depends on catalytically active Hel2 and on ribosomal protein Asc1. Lysates from log-phase cell cultures of the indicated strains in the S20-H 6 background without treatment (no-collision) or after a 10 min treatment with 1 µg/ml CHX (collision). Untagged wild type served as a control (ctrl). For details see Methods. Immunoblots were decorated with α-HIS, α-Hel2, α-Asc1, and α-Sse1 (loading control). The length of the ubiquitin chain attached to S20-H 6 is indicated; ubi 5+x indicates the smear of polyubiquitinated S20-H 6 species attached to more than 5 ubiquitin residues.

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Western Blot, Labeling, Concentration Assay, Control

Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: a Hel2 binds to translating ribosomes with low affinity. b A translating ribosome encounters a stalling-prone mRNA sequence. c A trailing ribosome catches up and collides with the stalled ribosome forming a disome. Hel2 binds with increased affinity to the disome and initiates ubiquitination of the 40S ribosomal protein Rps20. d The leading ribosome resumes translation, Hel2 reverts to its low-affinity binding mode, and ubiquitination of Rps20 ceases. e The deubiquitinases Ubp2 and Ubp3 remove ubiquitin chains from Rps20. This may occur during translation and/or after translation termination. f In case the leading ribosome stalls for a prolonged period of time, the disome develops the Asc1-Asc1 platform, which results in further increased affinity of Hel2. Hel2 now continuously extends the polyubiquitin chain until the RQC and NGD machineries are recruited and resolve the stalled ribosome. Hel2 in yellow, Asc1 in orange, Rps20 in white, ubiquitin moieties in red. For details see Discussion.

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Sequencing, Binding Assay