1. Volume 49, Issue 2, Pages 339-345 (January 2013)
The Human Base Excision Repair Enzyme SMUG1 Directly Interacts with DKC1 and Contributes to RNA Quality Control 
Laure Jobert, Hanne K. Skjeldam, Bjørn Dalhus, Anastasia Galashevskaya, Cathrine Broberg Vågbø, Magnar Bjørås, Hilde Nilsen 
Molecular Cell 
Volume 49, Issue 2, Pages (January 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1 SMUG1 Directly Interacts with DKC1
(A) HeLa cells expressing SMUG1-EYFP were treated or not with Actinomycin D, fixed, and stained with a DKC1-specific antibody and 4′,6-diamidino-2-phenylindole (DAPI). Confocal fluorescent images were obtained on a Zeiss LSM510 confocal microscope. Nucleoli and Cajal bodies are indicated by arrowheads and arrows, respectively.
(B) Coimmunoprecipitations were performed in cells expressing EYFP (lanes 1–6) or SMUG1-EYFP (lanes 7–12). Lysates were treated or not with DNase I or RNase A prior to immunoprecipitation. Coimmunoprecipitated proteins were detected by western blot analysis with EYFP- and DKC1-specific antibodies. IN, 10% input; IP, immunoprecipitate.
(C) Coimmunoprecipitations were performed with antibodies specific for DKC1 (lanes 2 and 3) or PCNA (lanes 4 and 5), as a negative control. Coimmunoprecipitated proteins were detected by western blot analysis with DKC1- and SMUG1-specific antibodies. IN, 10% input; IP, immunoprecipitate; IgG H, heavy chain of Immunoglobulin G.
(D) DKC1 model (blue) with the SMUG1-interacting peptide (amino acids 103–131, magenta). Amino acids used as restriction criteria are indicated.
(E) SMUG1 model (gold) with amino acids used as restriction criteria indicated.
(F) DKC1-SMUG1 docking model.
(G) Three-dimensional model of the interaction between SMUG1 and DKC1 together with the DKC1 partners GAR1 (purple), NOP-10 (yellow), and NHP2 (green).
(H) GST pull-downs were performed with recombinant purified GST or GST-DKC1 as baits and equivalent amounts of recombinant WT, E231R, or E29R E33R SMUG1 proteins. Results were analyzed by SDS-PAGE followed by western blotting analysis with the antibodies specific for GST and SMUG1. The bottom panel shows 5% input of each SMUG1 variant used for the GST pull-down.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 SMUG1 Has Activity on 5-hm(dUrd)-Containing ssRNA
(A) Activity of recombinant wt SMUG1 was assayed on a 25-mer single stranded and 5′-32P-end-labeled oligoribonucleotide substrate containing a centrally placed 5-hm(dUrd), Urd, or Ψ(Urd), as indicated. The substrates were incubated with no enzyme (–), UDG and APE1 (+), APE1 alone, or with increasing amounts of SMUG1 (10, 100, and 200 ng) and APE1. The 12-mer radiolabeled product was resolved by denaturing PAGE and detected by phosphorimager. S, substrate; P, product.
(B) Activity of recombinant WT and mutant H239L SMUG1 was assayed on 5-hm(dUrd)-containing ssRNA. Increasing amounts (1, 5, 10, 50, and 100 ng) of WT and mutant H239L SMUG1 were incubated with the 5′-32P-end-labeled substrate and APE1.
(C) SMUG1 excision activity (%) on 5-hm(dUrd)-containing ssRNA was calculated from three independent experiments and given as the mean ± SD.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 SMUG1 Contributes to rRNA Quality Control
(A) Native RNA coimmunoprecipitations were performed in cells overexpressing SMUG1-EYFP or EYFP alone. Reverse-transcription quantitative PCRs were performed with primers specific for the indicated RNAs. The data shown are the mean ± SEM from two independent experiments. Statistical significance was evaluated with the Student t test. ∗p < 0.02.
(B) After transfection with control or SMUG1 siRNAs for 48 hr, total RNA was purified and analyzed by reverse-transcription quantitative PCR with specific primers, as indicated. The data shown are the mean ± SEM from two independent experiments. Statistical significance was evaluated with the Student t test. ∗p < 0.005, ∗∗p < 0.05.
(C) Total and poly(A)+ RNA were prepared from cells transfected with control or SMUG1 siRNAs. Equal volumes of RNA samples were used as template in the RT reactions containing either random hexamers or oligo d(T) primers. Equal amounts of cDNAs were used in reverse-transcription quantitative PCR reactions with primers specific for the indicated RNAs. The results shown are the average of four PCRs from two independent RNA extractions. Statistical significance was evaluated using the Student t test. ∗∗p < 0.05.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
SMUG1 and DKC1 Prevent Accumulation of 5-hm(Urd) in 28S and 18S rRNAs In Vivo
(A) HeLa cells were transfected with control, SMUG1, and/or DKC1 siRNAs for 48 hr and whole-cell extracts were subjected to western blot analysis using DKC1-, GAPDH-, and SMUG1-specific antibodies.
(B and C) Quantification of incorporated Ψ(Urd) and 5-hm(Urd) per nucleotide RNA. Cells were harvested and 28S and 18S rRNA species were isolated, hydrolyzed, and analyzed for Ψ(Urd) or 5-hm(Urd) content by LC-MS/MS. Ψ(Urd) or 5-hm(Urd) levels are normalized relative to the total number of normal nucleosides measured. The data shown are the mean ± SD from three independent experiments.
(D–F) Cells expressing SMUG1-EYFP or SMUG1 E29R E33R-EYFP were fixed and stained with a DKC1-specific antibody and DAPI (D). Close-up of two neighboring cells from that do not (E) or do (F) express the SMUG1 E29R E33R-EYFP construct. Confocal fluorescent images were obtained by a Zeiss LSM510 confocal microscope. Nucleoli and Cajal bodies are indicated by arrowheads and arrows, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions