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Articles

Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay

Bernd Rattenbacher, Daniel Beisang, Darin L. Wiesner, Jonathan C. Jeschke, Maximilian von Hohenberg, Irina A. St. Louis-Vlasova, Paul R. Bohjanen
Bernd Rattenbacher
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
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Daniel Beisang
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
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Darin L. Wiesner
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
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Jonathan C. Jeschke
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
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Maximilian von Hohenberg
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
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Irina A. St. Louis-Vlasova
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
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Paul R. Bohjanen
1Center for Infectious Diseases and Microbiology Translational Research
2Department of Microbiology
3Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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  • For correspondence: Bohja001@umn.edu
DOI: 10.1128/MCB.00624-10
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  • FIG. 1.
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    FIG. 1.

    CUGBP1 selectively binds to GU-repeat sequences. (A) Sequences from the 3′ UTR of transcripts present in the anti-CUGBP1 IP were cloned into the 3′ UTR of the beta-globin reporter to create the JUNB-GRE (30), NDUFS2-GU, GSN-GU, and PPIC reporter transcripts. The indicated mutations and deletions were introduced to create the NDUFS2-mGU, NDUFS2-ΔGU, GSN-mGU, and GSN-ΔGU transcripts. (B) The GRE sequence of JUNB (JUNB-GRE) or the GU-repeat sequences of NDUFS2 (NDUFS2-GU), GSN (GSN-GU), or a mutated GU-repeat sequence from NDUFS2 (mGU) shown in the shaded boxes in panel A were used as radiolabeled RNA probes in EMSAs. Anti-CUGBP1 or anti-His antibodies were added to the indicated reaction mixtures. RNA-protein complexes were separated by electrophoresis under nondenaturing conditions. Gels were dried and analyzed on a phosphorimager. The positions of migration of the free probe, CUGBP1-containing band (CUGBP1), and the anti-CUGBP1 supershifted band (Supershift) are indicated.

  • FIG. 2.
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    FIG. 2.

    The GU-repeat sequence mediates rapid decay of the beta-globin reporter transcript. (A) HeLa Tet-Off cells were transfected with the pTetBBB beta-globin reporter construct or with reporter constructs in which GRE-containing sequences from the 3′ UTR of the JUNB transcript (JUNB-GRE) or GU-repeat sequences from the 3′ UTRs of the NDUFS2 (NDUFS2-GU), GSN (GSN-GU), or PPIC (PPIC-GU) were inserted into the beta-globin 3′ UTR. Doxycycline was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (B) The experiment shown in panel A was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.

  • FIG. 3.
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    FIG. 3.

    Mutation of the GU-repeat sequence abrogates mRNA decay. (A and B) The decay of the NDUFS2-GU (A) or the GSN-GU (B) beta-globin reporter was compared to the decay of these reporters in which the GU-repeats were deleted (NDUFS2-ΔGU and GSN-ΔGU) or mutated (NDUFS2-mGU and GSN-mGU). HeLa Tet-Off cells were transfected with the indicated beta-globin reporter constructs. Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C and D) The experiments shown in panels A and B were performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.

  • FIG. 4.
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    FIG. 4.

    CUGBP1 binding activity allows mismatches in the GU-repeat sequence. (A) Mutations and were introduced into the GSN-GU motif to create GM1 through GM8, as indicated. Mutated nucleotides are underlined. (B) The sequences depicted in panel A were used as radiolabeled RNA probes in EMSAs. The GSN-mGU sequence was used as a negative control. Anti-CUGBP1 or anti-His antibodies were added to the indicated reaction mixtures. RNA-protein complexes were separated by electrophoresis under nondenaturing conditions. Gels were dried and analyzed on a phosphorimager. The positions of migration of the free probe, CUGBP1-containing band (CUGBP1), and the anti-CUGBP1 supershifted band (Supershift) are indicated.

  • FIG. 5.
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    FIG. 5.

    CUGBP1 is responsible for GU-repeat-mediated mRNA decay. (A) The decay of the GSN-GU beta-globin reporter was measured in cells that expressed a control siRNA (Ctrl-si) or two different siRNAs (siA and siB) directed against CUGBP1. HeLa Tet-Off cells were transfected with the GSN-GU-repeat-containing reporter and the indicated siRNAs. Knockdown efficiency was monitored for each experiment by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. (B) Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at the 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C) The experiment shown in panel B was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are given in minutes.

  • FIG. 6.
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    FIG. 6.

    Generation of the GRE consensus sequence. Weblogo was generated by compiling a list of all occurrences of the GU repeat and the GRE found in the 3′ UTRs of those transcripts identified as present in the CUGBP1 IP. This list was uploaded into the program Weblogo, version 2.8.2, to generate the Weblogo (1).

  • FIG. 7.
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    FIG. 7.

    Posttranscriptional regulation of the CUGBP1 target network. (A) The network diagram depicts the coordinate regulation of CUGBP1 target transcripts involved in apoptosis. Transcripts depicted in orange represent GRE-containing transcripts that were enriched in the CUGBP1 targets. These pathways were identified by Ingenuity Pathway Assistant software (Ingenuity Systems, CA). (B) HeLa Tet-Off cells were transfected twice within 24 h with either a control siRNA or two different siRNAs specific for CUGBP1 (siA and siB). Knockdown efficiency was monitored by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. Apoptosis was assessed with a specific anti-PARP antibody. (C) Cells were stained for FACS analysis with a PE-labeled anti-active caspase 3 antibody. A representative experiment is shown. Four independent experiments were analyzed. Average percentages of apoptosis, standard error, and P values are indicated.

Tables

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  • Additional Files
  • TABLE 1.

    Subset of transcripts present in the anti-CUGBP1 IP

    RefSeq accession no.aGene name or descriptionGene symbolSequence(s)dP valuebGO biological function(s)c
    NM_000177Gelsolin (amyloidosis, Finnish type)GSNTGAGTGTGTGT, TGTGTGTGTGT0.003Actin filament polymerization, cell cycle
    NM_002229jun B proto-oncogeneJUNBTGTGTTTGTGT0.002Cell growth, cell cycle
    NM_004550NADH Dehydrogenase (ubiquinone) Fe-S protein 2NDUFS2TGTGTGTGTGT0.033Mitochondrial electron transport
    NM_000943Peptidylprolyl isomerase C (cyclophilin C)PPICTGTGTGTGTGT0.049Protein folding
    NM_005171Activating transcription factor 1ATF1TATGTGTGTGT, TGTGTGTGTGT0.015Cell growth, cell cycle
    NM_005354jun D proto-oncogeneJUNDTGTGTGTGTGT0.0005Cell growth, cell cycle
    NM_001769CD9 moleculeCD9TTTTTGTTTGT, TGTTTGTTTTT0.0001Motility, cell growth
    NM_014989Regulating synaptic membrane exocytosis 1RIMS1TGTTTATTTGT, TGTTTGTTGGT0.0017Protein complex assembly
    NM_012197Rab GTPase activating protein 1RABGAP1TGTGTGTGTGT0.008Cell cycle
    NM_004217Aurora kinase BAURKBTGTTTGTATGT0.045Cytokinesis, cell cycle
    NM_000641Interleukin-11IL-11TGTTTGTTTTT0.050Cell-cell signaling
    NM_018685Anillin, actin binding proteinANLNTGTTTGTTTGT0.043Cytokinesis
    NM_001894Casein kinase 1, epsilonCSNK1ETGTGTGTGTGT0.010DNA repair
    NM_001080421unc-13 homolog A (C. elegans)UNC13ATGTGTGTGTGT, TGTTTGTTTTT0.041Exocytosis
    NM_015083Cdc2-related kinase, arginine/serine-richCRKRSTGTGTGTGTGT0.044Protein amino acid phosphorylation
    NM_002578p21 (CDKN1A)-activated kinase 3PAK3TGTTTGTTTTT0.009Protein complex assembly
    NM_001006610Seven in absentia homolog 1 (Drosophila)SIAH1TGTGTGCGTGT0.007Proteolysis
    NM_002468Myeloid differentiation primary response gene 88MYD88TGGGTGTGTGT, GGTGTGTGTGT0.022Response to molecule of fungal origin
    NM_005633Son of sevenless homolog 1 (Drosophila)SOS1TGTTTGTGTAT0.042Signal transduction
    • ↵ a From the NCBI Reference Sequence Project.

    • ↵ b P value indicates the statistical significance of the enrichment of the transcript in the anti-CUGBP1 IP compared to the anti-HA IP.

    • ↵ c GO, gene ontology.

    • ↵ d Some transcripts contain more than one enriched GU-rich sequence.

  • TABLE 2.

    Prevalence of motifs in the 3′ UTR of transcripts in the anti-CUGBP1 compared to all transcripts in the genome

    MotifPrevalence in the anti-CUGBP1 IP (%)Prevalence in the genome (%)P value
    TGTGTGTGTGT17.337.62.53 × 10−15
    GGAGGAGGAGG8.515.41.02 × 10−2
    TGTTTGTTTGT18.696.75.35 × 10−26
    TTTTTTTTTTT38.2821.88.03 × 10−8
    TGGGAATGGTC2.580.86.28 × 10−6
    CACACACACAC7.754.91.40 × 10−3

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Table S1 (Transcripts present in anti-CUGBP1 IP)
      Zipped MS Excel file, 46K.
    • Supplemental file 2 - Fig. S1 (GRE and GU-repeat sequences and reporter translation)
      Zipped PDF file, 1.5 MB.
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Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay
Bernd Rattenbacher, Daniel Beisang, Darin L. Wiesner, Jonathan C. Jeschke, Maximilian von Hohenberg, Irina A. St. Louis-Vlasova, Paul R. Bohjanen
Molecular and Cellular Biology Jul 2010, 30 (16) 3970-3980; DOI: 10.1128/MCB.00624-10

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Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay
Bernd Rattenbacher, Daniel Beisang, Darin L. Wiesner, Jonathan C. Jeschke, Maximilian von Hohenberg, Irina A. St. Louis-Vlasova, Paul R. Bohjanen
Molecular and Cellular Biology Jul 2010, 30 (16) 3970-3980; DOI: 10.1128/MCB.00624-10
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KEYWORDS

RNA, Messenger
RNA-binding proteins

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