Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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.
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
- TABLE 1.
Subset of transcripts present in the anti-CUGBP1 IP
RefSeq accession no.a Gene name or description Gene symbol Sequence(s)d P valueb GO biological function(s)c NM_000177 Gelsolin (amyloidosis, Finnish type) GSN TGAGTGTGTGT, TGTGTGTGTGT 0.003 Actin filament polymerization, cell cycle NM_002229 jun B proto-oncogene JUNB TGTGTTTGTGT 0.002 Cell growth, cell cycle NM_004550 NADH Dehydrogenase (ubiquinone) Fe-S protein 2 NDUFS2 TGTGTGTGTGT 0.033 Mitochondrial electron transport NM_000943 Peptidylprolyl isomerase C (cyclophilin C) PPIC TGTGTGTGTGT 0.049 Protein folding NM_005171 Activating transcription factor 1 ATF1 TATGTGTGTGT, TGTGTGTGTGT 0.015 Cell growth, cell cycle NM_005354 jun D proto-oncogene JUND TGTGTGTGTGT 0.0005 Cell growth, cell cycle NM_001769 CD9 molecule CD9 TTTTTGTTTGT, TGTTTGTTTTT 0.0001 Motility, cell growth NM_014989 Regulating synaptic membrane exocytosis 1 RIMS1 TGTTTATTTGT, TGTTTGTTGGT 0.0017 Protein complex assembly NM_012197 Rab GTPase activating protein 1 RABGAP1 TGTGTGTGTGT 0.008 Cell cycle NM_004217 Aurora kinase B AURKB TGTTTGTATGT 0.045 Cytokinesis, cell cycle NM_000641 Interleukin-11 IL-11 TGTTTGTTTTT 0.050 Cell-cell signaling NM_018685 Anillin, actin binding protein ANLN TGTTTGTTTGT 0.043 Cytokinesis NM_001894 Casein kinase 1, epsilon CSNK1E TGTGTGTGTGT 0.010 DNA repair NM_001080421 unc-13 homolog A (C. elegans) UNC13A TGTGTGTGTGT, TGTTTGTTTTT 0.041 Exocytosis NM_015083 Cdc2-related kinase, arginine/serine-rich CRKRS TGTGTGTGTGT 0.044 Protein amino acid phosphorylation NM_002578 p21 (CDKN1A)-activated kinase 3 PAK3 TGTTTGTTTTT 0.009 Protein complex assembly NM_001006610 Seven in absentia homolog 1 (Drosophila) SIAH1 TGTGTGCGTGT 0.007 Proteolysis NM_002468 Myeloid differentiation primary response gene 88 MYD88 TGGGTGTGTGT, GGTGTGTGTGT 0.022 Response to molecule of fungal origin NM_005633 Son of sevenless homolog 1 (Drosophila) SOS1 TGTTTGTGTAT 0.042 Signal transduction - TABLE 2.
Prevalence of motifs in the 3′ UTR of transcripts in the anti-CUGBP1 compared to all transcripts in the genome
Motif Prevalence in the anti-CUGBP1 IP (%) Prevalence in the genome (%) P value TGTGTGTGTGT 17.33 7.6 2.53 × 10−15 GGAGGAGGAGG 8.51 5.4 1.02 × 10−2 TGTTTGTTTGT 18.69 6.7 5.35 × 10−26 TTTTTTTTTTT 38.28 21.8 8.03 × 10−8 TGGGAATGGTC 2.58 0.8 6.28 × 10−6 CACACACACAC 7.75 4.9 1.40 × 10−3
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1 (Transcripts present in anti-CUGBP1 IP)
Zipped MS Excel file, 46K. - Supplemental file 2
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Fig. S1 (GRE and GU-repeat sequences and reporter translation)
Zipped PDF file, 1.5 MB.
- Supplemental file 1
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Table S1 (Transcripts present in anti-CUGBP1 IP)
