Conserved GU-Rich Elements Mediate mRNA Decay by Binding to CUG-Binding Protein 1

Conserved GU-Rich Elements Mediate mRNA Decay by Binding toCUG-Binding Protein 1

Irina A. Vlasova1,2, Nuzha M. Tahoe2, Danhua Fan3, Ola Larsson3, Bernd Rattenbacher1,2,Julius R. SternJohn1,2, Jayprakash Vasdewani2,4, George Karypis4, Cavan S. Reilly5, PeterB. Bitterman3, and Paul R. Bohjanen1,2,3,*

1Center for Infectious Diseases and Microbiology Translational Research University of Minnesota,Minneapolis, MN 55455, USA

2Department of Microbiology University of Minnesota, Minneapolis, MN 55455, USA

3Department of Medicine University of Minnesota, Minneapolis, MN 55455, USA

4Department of Computer Science University of Minnesota, Minneapolis, MN 55455, USA

5Division of Biostatistics University of Minnesota, Minneapolis, MN 55455, USA

SUMMARYWe used computational algorithms to find conserved sequences in the 3′ untranslated region (UTR)of transcripts that exhibited rapid decay in primary human T cells and found that the consensussequence UGUUUGUUUGU, which we have termed a GU-rich element (GRE), was enriched inshort-lived transcripts. Using a tet-off reporter system, we showed that insertion of GRE-containingsequences from c-jun, jun B, or TNF receptor 1B, but not mutated GRE sequences, into the 3′UTRof a β-globin transcript conferred instability on the otherwise stable β-globin transcript. CUG-bindingprotein 1 (CUGBP1) was identified as the major GRE-binding activity in cytoplasmic extracts fromprimary human T cells based on supershift and immunoprecipitation assays. siRNA-mediatedknockdown of CUGBP1 in HeLa cells caused stabilization of GRE-containing transcripts, suggestingthat CUGBP1 is a mediator of GRE-dependent mRNA decay. Overall, our results suggest that theGRE mediates coordinated mRNA decay by binding to CUGBP1.

INTRODUCTIONThe integration of signals from multiple regulatory proteins that bind to mRNA coordinatelyregulates selective mRNA decay during development and in response to environmental stimuli(Keene, 2007; Raghavan and Bohjanen, 2004). The best characterized example of coordinategene regulation through selective mRNA decay is the regulation of mRNA decay by AU-richelements (AREs). AREs are conserved sequences found in the 3′ untranslated region (UTR)from a variety of short-lived transcripts, including cytokine and proto-oncogene transcripts,which function to mediate rapid mRNA decay (Ogilvie et al., 2005; Raghavan et al., 2004).AREs mediate mRNA decay by interacting with ARE-binding proteins, such as K homologysplicing regulatory protein (KSRP), tristetraprolin (TTP), and butyrate response factor 1(BRF-1), that recruit components of the mRNA decay machinery to specific transcripts (Chenet al., 2001; Gherzi et al., 2004; Hau et al., 2007; Lykke-Andersen and Wagner, 2005), therebycoordinately regulating mRNA decay.

*Correspondence: bohja001@umn.eduSupplemental DataSupplemental Data include two figures and one table and can be found with this article online athttp://www.molecule.org/cgi/content/full/29/2/263/DC1/.

NIH Public AccessAuthor ManuscriptMol Cell. Author manuscript; available in PMC 2008 May 5.

Published in final edited form as:Mol Cell. 2008 February 1; 29(2): 263–270.

-PA Author Manuscript

We have previously used microarrays to measure mRNA decay on a genome-wide scale inprimary human T cells and identified hundreds of transcripts that exhibited rapid decay(Raghavan et al., 2002; Vlasova et al., 2005). Most of these short-lived transcripts did notcontain AREs or other known RNA regulatory elements (Raghavan et al., 2004), leading us tohypothesize that they may contain other regulatory sequences that would allow them to beselectively recognized. In this work, we used computational methods to determine that theconserved 11-mer sequence UGUUUGUUUGU, which we have termed the GU-rich element(GRE), was enriched in the 3′UTRs of short-lived transcripts. Introduction of GRE sequencesinto the 3′UTR induced destabilization of an otherwise stable β-globin reporter transcript,demonstrating that the GRE is a functional mediator of mRNA decay. The RNA-bindingprotein CUG-binding protein 1 (CUGBP1), the mammalian homolog of the deadenylationregulator EDEN-BP in Xenopus, bound specifically to the GRE and mediated GRE-dependentmRNA decay. The GRE and CUGBP1 are components of a selective mRNA decay pathwaythat regulates the expression of an important subset of human transcripts.

RESULTS AND DISCUSSIONBecause most transcripts that are regulated at the level of mRNA decay do not contain AREsor other known sequence elements that regulate mRNA decay (Raghavan et al., 2002, 2004),we hypothesized the existence of other regulatory elements that had not yet been discovered.We used computational methods to search for conserved oligomer sequences that wereenriched within short-lived transcripts which were induced following T cell activation (see theExperimental Procedures) and found that the 11-mer consensus sequence UGUUUGUUUGU,which we have termed a GRE, was enriched in the 3′UTR of short-lived transcripts. Weretrieved the 3′UTR sequences from 4812 transcripts for which we had measured mRNA half-lives in T cells (Raghavan et al., 2002), and searched within these 3′UTR sequences for the11-mer consensus GRE, allowing one mismatch. We then compared the abundance of thisGRE 11-mer sequence in the set of 384 transcripts that exhibited very rapid decay (upper limitof half-life 95% confidence interval of less than 60 min) to the set of 1795 transcripts that werevery stable (lower limit of the half-life 95% confidence interval of greater than 360 min), andfound significant enrichment (p < 0.0001) of the GRE in short-lived transcripts, with 11.98%of short-lived and 5.79% of long-lived transcripts containing this sequence. As a positivecontrol, we evaluated the abundance of AREs in the 3′UTR of these sets of transcripts andfound significant enrichment in short-lived transcripts with 9.90% of short-lived and 5.91% oflong-lived transcripts containing a class I ARE (p < 0.01) and 5.21% of short-lived and 2.51%of long-lived transcripts containing a class II ARE (p < 0.01). Thus, the GRE exhibited a similardegree of abundance and enrichment in short-lived transcripts as these AREs. Overall, wefound that the 11-mer GRE was present in ∼5% of the 4812 transcripts expressed in T cellsfor which we had measured mRNA decay rates. A list of these GRE-containing transcripts andtheir mRNA half-lives is included in Table S1 available online. The 11-mer GRE sequencewas present in the 3′UTRs of ∼100 short-lived transcripts with half-lives of less than 60 min.A subset of these transcripts is listed in Table 1. Many of these GRE-containing transcriptsencode important regulatory proteins in the cell, including transcription factors, proto-oncogenes, apoptosis regulators, signal transduction regulators, and regulators of metabolism.

The finding that the GRE is more abundant in short-lived transcripts compared to stabletranscripts led us to hypothesize that this conserved sequence may function as a mediator ofmRNA decay. To test this hypothesis, HeLa Tet-off cells were transiently transfected with tet-repressible β-globin reporter constructs in which the GRE-containing sequences from c-jun,jun B, or TNF receptor superfamily member 1B (TNFRSF1B) shown in Figure 1A wereinserted into the 3′UTR. The c-jun GRE sequence was chosen for these experiments becauseit was contained within a previously characterized 150 nucleotide functional decay elementthat was classified as a class III ARE based on its overall AU richness (Peng et al., 1996). A

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green fluorescence protein (GFP) expression construct that was not tetracycline regulated wascotransfected along with the tetracycline-repressible β-globin reporter constructs to control fortransfection efficiency. After 48 hr, doxycycline was added to the medium to stop transcriptionfrom the tet-responsive promoter and then β-globin and GFP mRNA levels were measuredover time by northern blot (Figures 1B and 1C). In the absence of an insert, the β-globintranscript was very stable with a half-life of 3188 ± 200 min (mean ± standard error of themean). Insertion of the GRE-containing sequences from c-jun, jun B, and TNFRSF1B led toaccelerated decay of the β-globin reporter with half-lives of 189 ± 53 min, 200 ± 50 min, and196 ± 34 min, respectively. In contrast, insertion of the mutated sequences into 3′UTR hadrelatively little effect on mRNA decay rates with a half-life of 1031 ± 298 min, 1019 ± 230min, and 1689 ± 542 min for the mutated c-jun, jun B, and TNFRSF1B GRE, respectively.Thus, the introduction of short GRE-containing sequences into the 3′UTR of β-globin mRNAis sufficient for enhancing transcript decay, suggesting that the GRE is a functional mediatorof mRNA decay.

Because AREs and other RNA elements that regulate mRNA decay function through theirinteractions with specific RNA-binding proteins, we hypothesized that the GRE may alsofunction by interacting with specific RNA-binding proteins. We performed gel shift assaysusing cytoplasmic extracts from primary human T cells to search for GRE-specific RNA-binding activities (Figure 2A). A 22 nucleotide radiolabeled ribo-oligonucleotide probe thatcontained the boxed c-jun GRE sequence shown in Figure 1A was mixed with T cellcytoplasmic extracts, and the mixtures were separated by electrophoresis on a nondenaturingpolyacrylamide gel. A major c-jun GRE-binding activity was observed (lane 1), and bindingby this activity was competed by the addition of excess of unlabeled ribo-oligonucleotides thatcontained the boxed c-jun (lane 2), jun B (lane 4), and TNFRSF1B (lane 6) GRE sequencesshown in Figure 1A, but not by the indicated mutated sequences (lanes 3, 5, and 7). Bindingby this activity was also not competed by unlabeled ribo-oligonucleotides containing a 22nucleotide GM-CSF ARE sequence or by a 22 nucleotide poly U sequence (data not shown).These data suggested that the GRE-binding activity was sequence specific.

In order to identify this RNA-binding activity, we performed supershift assays using antibodiesagainst RNA-binding proteins that have specificity for U-rich or GU-rich sequences, includingCUGBP1, CUGBP2, HuR, TTP, and KSRP. Supershift assays (Figure 2B) were performedusing T cell cytoplasmic extracts and radiolabeled GRE or mutated GRE sequences from c-jun, jun B, and TNFRSF1B, and we found that RNA-binding complexes that bound to the GREsequences were specifically supershifted with an anti-CUGBP1 antibody (lanes 2, 8, and 14),suggesting that they contained CUGBP1. In contrast, complexes that bound to the mutatedsequences were not super-shifted with anti-CUGBP1 antibodies (lanes 5, 11, and 17),suggesting that they did not contain CUGBP1 but were composed of other cellular proteins.Antibodies against actin (lanes 3, 9, and 15) or against other RNA-binding proteins includingHuR, TTP, CUGBP2, and KSRP did not supershift the GRE-containing complexes (data notshown). Using an RNA-protein UV crosslinking assay we identified a major RNA-bindingactivity that recognized the c-jun GRE sequence and had a molecular weight of ∼55 kDa, andbinding by this activity was competed by the addition of excess unlabeled c-jun, jun B, orTNFRSF1B GRE ribo-oligonucleotides but was not competed by mutated oligonucleotides(Figure S1A), suggesting that this 55 kDa RNA-binding activity specifically recognizes GREsequences. An anti-CUGBP1 antibody specifically immunoprecipitated the GRE-specific 55kDa RNA-protein complex (Figure S1B), suggesting that this complex contains CUGBP1.Overall, these results indicate that the major GRE-binding activity that we identified in T cellcytoplasmic extracts is CUGBP1.

CUGBP1, a member of the CELF family of RNA-binding proteins, was first described as aprotein that bound to the abnormally extended CUG mRNA repeats that occur in patients with

type I myotonic dystrophy (Timchenko et al., 1996) and has since been implicated as a regulatorof translation (Timchenko et al., 1999) and alternative splicing (Philips et al., 1998). A searchfor preferential RNA-binding sites using systemic evolution of ligands exponential enrichment(SELEX) revealed that CUGBP1 bound to UGUU-rich sequences with high affinity (Marquiset al., 2006). We performed competitive RNA-binding experiments and determined theapparent affinity of the CUGBP1-containing complex for the c-jun GRE to be 5.2 ± 1.3 nM(Figures 2C and 2D). We also produced recombinant CUGBP1 in E. coli and tested itsspecificity for binding to GRE sequences (Figure S1C). Binding by recombinant CUGBP1 toradiolabeled c-jun GRE RNA was competed by the addition of increasing amounts of unlabeledc-jun RNA, but not mutated c-jun RNA, indicating that CUGBP1 binding to the GRE isspecific. Based on competitive binding results, we calculated the affinity of recombinantCUGBP1 for the c-jun GRE sequence to be 10.6 ± 2.7 nM. This affinity is similar to thepreviously reported affinity of CUGBP1 for artificially selected GU-rich sequences (Marquiset al., 2006), and is similar to the affinity of functional ARE-binding proteins for their targetsequences (Hau et al., 2007). Overall, these data demonstrate that CUGBP1 binds specificallyto GRE sequences with high affinity.

Because CUGBP1 bound specifically to short GRE sequences in vitro, we performed RNAimmunoprecipitation assays to determine whether CUGBP1 also bound specifically to GRE-containing full-length reporter transcripts expressed in cells. HeLa Tet-off cells weretransiently transfected with a β-globin reporter construct or β-globin reporter constructs inwhich the TNFRSF1B GRE or a mutated sequence was inserted into its 3′UTR. Lysates fromthese cells were immunoprecipitated using an anti-CUGBP1 antibody, an antibody againstpoly(A)-binding protein (PABP), or an anti-HA antibody. RNA was isolated from theimmunoprecipitated material and reverse transcriptase-polymerase chain reaction (RT-PCR)was performed to detect β-globin or GAPDH transcripts. As seen in Figure 2E, the anti-CUGBP1 antibody immunoprecipitated the β-globin transcript only when the GRE insert waspresent (lane 16), but not when no insert (lane 4) or a mutated GRE insert (lane 10) was present.In contrast, an anti-PABP antibody immunoprecipitated the β-globin transcripts regardless ofwhether or not a GRE insert was present (lanes 6, 12, and 18). The anti-PABP antibody alsoimmunoprecipitated endogenous GAPDH transcripts (lanes 6, 12, and 18), whereas the anti-CUGBP1 antibody did not (lanes 4, 10, and 16). The anti-HA antibody, which served as anegative control, did not effectively immunoprecipitate either transcript (lanes 2, 8, and 14).These results suggest that CUGBP1 is capable of selectively recognizing GRE-containingtranscripts in cells.

Based on our findings that the GRE functions to mediate mRNA decay and that CUGBP1 bindsto the GRE with high affinity, we hypothesized that CUGBP1 functions as a mediator of mRNAdecay. CUGBP1 is evolutionarily conserved, and its homolog in Xenopus, embryodeadenylation element-binding protein (EDEN-BP), serves dual functions as a translationalrepressor in oocytes and a deadenylation factor following fertilization (Paillard et al., 1998).EDEN-BP regulates deadenylation by binding to the embryonic deadenylation element(EDEN), a sequence that is rich in uridine and purine dinucleotides (Paillard et al., 1998).Interestingly, CUGBP1 was able to functionally substitute for EDEN-BP to mediate transcriptdeadenylation in Xenopus extracts (Paillard et al., 2003), suggesting that it may play a role intranscript deadenylation. Tethering of CUGBP1 to the 3′UTR of mRNA through an interactionwith the MS2 coat protein led to decreased steady-state levels of reporter transcripts thatcontained an MS2 RNA-binding site while reporter protein levels increased (Barreau et al.,2006), suggesting that CUGBP1 could play a dual role in translation and/or mRNA decay.Recently, CUGBP1 was shown to associate with poly(A) ribonuclease and to stimulate poly(A) tail shortening in HeLa cell S100 extracts (Moraes et al., 2006), suggesting that CUGBP1may play a role in transcript deadenylation.

To determine whether CUGBP1 is a regulator of GRE-mediated mRNA decay, we evaluatedthe effect of siRNA-mediated knockdown of CUGBP1 on the decay of GRE-containingreporter transcripts. HeLa tet-off cells were treated with two independent siRNAs (siCUGBP1-A and siCUGBP1-B) designed to target CUGBP1 or a control siRNA that targets redfluorescence protein. Both of the siRNAs directed against CUGBP1 or a pool of siRNAs againstCUGBP1 led to a decrease in CUGBP1 protein level by 80%–99% as assessed by western blot(see Figure S1). These same cells were used to assess the decay of β-globin reporter transcriptsthat contained a GRE insertion (Figures 3A and 3B). In cells that were transfected with the redfluorescence protein control siRNA, the decay rate of the β-globin reporter transcript thatcontained the TNFRSF1B GRE insertion was 183 ± 159 min. In contrast, this transcript wasstabilized (p < 0.05) in cells treated with siRNAs directed against CUGBP1 with half-lives of1689 ± 635 min or 1102 ± 202 min following treatment with siCUGBP1-A or siCUGBP1-B,respectively. We found similar results when we used a pool of siRNAs directed againstCUGBP1 (Figures 3C and 3D). In cells that were transfected with a pool of nontargetingsiRNAs, the decay rates of β-globin reporters that contained TNFRSF1B GRE and jun B GREinsertions were 151 ± 37 min and 193 ± 22 min, respectively. These transcripts were stabilized(p < 0.05) in cells treated with siRNAs directed against CUGBP1 with a half-life of theTNFRSF1B GRE-containing transcript of 451 ± 70 min and a half-life of the jun B GRE-containing transcript of 708 ± 56 min. These findings suggest that CUGBP1 is a mediator ofGRE-directed mRNA decay and that insertion of a GRE into the 3′UTR of the β-globintranscript is sufficient to allow CUGBP1 to function.

The RNA immunoprecipitation assay was also performed to determine whether CUGBP1 wasable to bind to endogenous GRE-containing transcripts in cells. HeLa cell lysates wereimmunoprecipitated with an anti-CUGBP1 antibody or an anti-HA antibody, and RT-PCR wasused to assess the presence of endogenous c-jun, jun B, or GAPDH transcripts in RNA isolatedfrom the immunoprecipitated material (Figure 3E). The anti-CUGBP1 antibodyimmunoprecipitated c-jun and jun B transcripts, but not the GAPDH transcripts, whereas theanti-HA antibody did not effectively immunoprecipitate any of these transcripts. These resultssuggest that CUGBP1 was able to bind to endogenous GRE-containing transcripts in cells. Todetermine the functional consequences of CUGBP1 binding to endogenous RNA in cells, wealso evaluated the effect of siRNA-mediated knockdown of CUGBP1 on the decay ofendogenous c-jun and jun B transcripts. HeLa cells were treated with siRNA directed againstCUGBP1 or control siRNA, then actinomycin D was added to stop transcription by RNApolymerase II, and the decay of c-jun and jun B transcripts was measured using real-time RT-PCR (Figure 3F). siRNA-mediated knockdown of CUGBP1 led to the stabilization of thesetranscripts, suggesting that in addition to mediating the decay of GRE-containing reportertranscripts, CUGBP1 also mediates the decay of endogenous GRE-containing transcripts.

Overall, we have identified the GRE as an mRNA decay element and have defined CUGBP1as a functional mediator of GRE-dependent mRNA decay. Our results demonstrate thatCUGBP1 is a regulator of mammalian gene expression at the level of mRNA decay and thatCUGBP1 and the GRE define a conserved pathway that selectively regulates mRNA decay.

EXPERIMENTAL PROCEDURESIdentification and Analysis of Conserved GU-Rich Sequences

We previously used microarrays to measure the mRNA decay rates of ∼6000 transcriptsexpressed in resting and activated primary human T cells (Raghavan et al., 2002; Vlasova etal., 2005). We analyzed the set of transcripts that were induced greater than 2-fold (p ≤ 0.05)following stimulation of primary human T cells with anti-CD3 and anti-CD28 antibodies anddivided these transcripts into two groups: one group contained transcripts with half-lives lessthan or equal to 60 min and the other group contained transcripts with half-lives greater than

360 min. We examined the frequencies of all possible 12-mer sequences (412) within the 3′UTR of these transcripts and compiled the top 1000 12-mers that had a higher frequency in theshort-lived transcripts compared to the long-lived transcripts. Using a 1000×1000 similaritymatrix, we performed agglomerative clustering using the program CLUTO (Rasmussen et al.,2003). The similarity matrix was computed simply by calculating the alignment score betweentwo 12-mers. The match was scored as 1 and mismatch 0, and no gaps were allowed. CLUTOoutput provided for each cluster, the number of objects, average similarity between the objectsof each cluster (internal similarity), and the average similarity of the objects of each clusterand the rest of the objects (external similarity). A promising cluster contained the sequenceUGUUUGUUUGUU.

Further evaluation of all short-lived transcripts expressed in primary human T cells revealedthat the related 11-mer sequence UGUUUGUUUGU occurred frequently in the 3′UTR of thesetranscripts, and therefore this conserved sequence was analyzed in more detail. Sequences andannotations, including the positions of the 3′UTRs, for 4812 transcripts for which we hadmeasured mRNA decay rates were retrieved from the RefSeq database, and the 3′UTRsequences were extracted using scripts written in the Practical Extraction and Report Language(PERL). FindPatterns from GCG (Version 11.0, Accelrys, San Diego) was used to search forthe 11-mer sequence UGUUUGUUUGU, permitting one mismatch. Transcripts containing anARE were extracted from the ARE 3.0 database (Bakheet et al., 2006). ARE-containingtranscripts were further separated into transcripts containing class I or class II AREs usingPERL scripts. We compared the abundance of the GU-rich 11-mer sequence or the AREsequences in short-lived transcripts defined as transcripts whose half-life 95% confidenceinterval upper limit was less than 60 min to their abundance in long-lived transcripts definedas transcripts whose half-life 95% confidence interval lower limit was greater than 360 min.Enrichments were tested for significance using Fisher's exact test in R(http://www.r-project.org).

Tet-Off mRNA Decay AssayThe decay of β-globin reporter constructs was performed as described previously (Ogilvie etal., 2005), with minor modifications. HeLa tet-off cells (1.6 × 106 cells) were transfected with3.0 μg of the tet-responsive reporter construct that encoded the rabbit β-globin transcript orrabbit β-globin transcripts that contained the additional sequences shown in Figure 1A insertedat the unique BglII restriction site in its 3′UTR. Tet-responsive rabbit β-globin constructs,pTetBBB and pTetBBB/c-jun (Peng et al., 1996), were gifts from Dr. Ann-Bin Shyu(University of Texas-Houston). The c-jun, mutated c-jun (mc-jun), jun B, mutated jun B (mjunB), TNFRSF1B, and mutated TNFRSF1B (mTNFRSF1B) sequences were inserted into thepTetBBB plasmid using the GeneTailor site-directed mutagenesis system (Invitrogen,Carlsbad, CA). The HeLa tet-off cells were cotransfected with 1 μg of the pTracerC-EF/V5-His/lacZ construct (Invitrogen Life Technologies), which produces GFP, to control fortransfection efficiency. Transfections were performed with 2.5 U of TransIT-LT1 reagent(Mirus, Madison, WI) per microgram of plasmid DNA. After 48 hr, 300 ng/ml of doxycyclinewas added to stop transcription, and total RNA was isolated after 0, 2, 4, and 6 hr. RNA isolationand northern blotting to assess expression of β-globin and GFP transcripts were performed asdescribed previously (Ogilvie et al., 2005). For each point, the hybridization intensity of theβ-globin transcript was normalized to the hybridization intensity of the GFP transcript, and thenormalized values were used to calculate half-lives using GraphPad Prism 4.03 software basedon a one-phase exponential model of decay.

Gel Shift, Supershift, and UV-Crosslinking AssaysRibo-oligonucleotides were purchased commercially (Dharmacon Research, Boulder, CO).The sequences for each ribo-oligonucleotide are shown as the boxed sequences in Figure 1A.

Radiolabeled ribo-oligonucleotide probes were end-labeled with [γ-32P]ATP (6000 Ci/mmol)using T4 polynucleotide kinase (Invitrogen) to produce a radiolabeled probe with a specificactivity of 4 × 106 cpm/μg. Gel shift and UV crosslinking assays were conducted as describedpreviously (Raghavan et al., 2001). Each reaction contained 8–10 μg of cytoplasmic proteinand 10–15 fmol of radiolabeled RNA probe in a total volume of 20–24 μl. For supershift assays,an anti-CUGBP1 mouse monoclonal antibody (3B1, Santa Cruz Biotechnologies) or an anti-actin mouse monoclonal antibody (C-2, Santa Cruz Biotechnologies) was added to the reactionmixtures. The gels were dried and analyzed on a phosphorimager (Molecular Dynamics). Forcompetitive binding assays, GraphPad Prism version 4.03 (GraphPad Software, San Diego,CA) was used to graph binding data and calculate apparent affinities and standard errors usinga homologous competition with depletion one-site binding model.

RNA Immunoprecipitation AssaysRNA immunoprecipitation assays were performed as described (Tenenbaum et al., 2002). RNAwas extracted from the immunoprecipitated samples and the input samples using the RNeasykit (QIAGEN) following the manufacturer's protocol, including a DNase digest. SuperscriptII reverse transcriptase (Invitrogen) was used to convert 4 μg of total RNA or a correspondingamount of immunoprecipitated RNA using an oligo dT15 primer. Products were amplified for38 cycles from cDNA using the following primers: β-globin forward 5′-GTCTACCCATGGACCCAGAGG-3′, β-globin reverse 5′-GTGAGCGGCATTGGCCACACC-3′;c-jun forward 5′-CCCCAAGATCCTGAAACAGA-3′,c-jun reverse 5′-CCGTTGCTGGACTGGATTAT-3′; jun B forward 5′-TGGAACAGCCCTTC TACCAC-3′,jun B reverse 5′-CTCAGGAGCATGGGGATAAA-3′; GAPDH forward 5′-TGATGGTACATGACAAGGTGC-3′, GAPDH reverse 5′-ACAGTCCATGCCATCACTGC-3′.

siRNA TransfectionThe following siRNA duplexes were purchased commercially from Dharmacon Research(Boulder, CO): siCUGBP1-A (5′-GAGCCAACCUGUUCAUCUA-3′; catalog numberJ-020166-05), siCUGBP1-B (5′-GCUGUUUAUUGGUAUGA UU-3′; catalog numberD-020166-04), siRNA targeting red fluorescence protein (5′-AAAGGACGGAGGACATTAT-3′), smart pool siRNAs representing a mixture of fourdistinct RNA duplexes directed against CUGBP1 (catalog number M-020166), and a controlpool of nontargeting siRNAs. For the Tet-off mRNA decay reporter assay, HeLa tet-off cells(4 × 105 cells) were transfected sequentially two or three times at 24 hr intervals with 50–100nmol of siRNAs directed against CUGBP1 or control siRNAs according to the manufacturer'sinstructions. For Tet-off mRNA decay assays, 24 hr after the last siRNA transfection, the cellswere then transfected with β-globin and GFP constructs and mRNA decay was measured asdescribed above. For actinomycin D mRNA decay assays, actinomycin D (10 ug/ml) was addedto the culture media, and total RNA harvested after 0, 1, and 2 hr was analyzed by real-timeRT-PCR using transcript-specific primers to evaluate mRNA decay as described previously(Vlasova et al., 2005). In each experiment, aliquots of siRNA-transfected cells were used toprepare total cellular extracts (Atasoy et al., 2003) for western blotting with an anti-CUGBP1mouse monoclonal antibody (Santa Cruz Biotechnologies) or an anti-actin mouse monoclonalantibody (Calbiochem) to assess the effectiveness of CUGBP1 knockdown.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

ACKNOWLEDGMENTS

This work was supported by grants AI49494 and AI52170 from the NIH to P.R.B. and by a research grant from theMinnesota Medical Foundation (to P.R.B. and I.A.V.). I.A.V. was funded through awards from the MinnesotaSupercomputing Institute and the Lymphoma Research Foundation. N.M.T. was funded by the Pediatric InfectiousDiseases Training Program (NIH grant T32 HD007381). O.L. was supported by a postdoctoral fellowship from theSwedish Research Council. D.F. and P.B.B. were supported by HL076779 and HL073719 from the NIH. We thankDr. Ann-Bin Shyu and Dr. Thomas Cooper for providing plasmids.

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Figure 1. GREs Are Functional Mediators of mRNA Decay(A) The shown GRE-containing sequences from the 3′UTR of c-jun, jun B, and TNFRSF1Bwere cloned into the 3′UTR of the pTetBBB β-globin reporter construct. The boxed sequencesindicate the sequences of the ribo-oligonucleotides used for the binding reactions shown inFigure 2. The arrows indicate the single-nucleotide mutations (G to C) that were introduced tocreate the mutated GRE sequences.(B) HeLa Tet-off cells were transfected with the pTracer GFP expression construct and thepTetBBB β-globin reporter construct (No Insert) or with reporter constructs in which GRE-containing sequences (c-jun, jun B, and TNFRSF1B) or mutated sequences (mc-jun, mjun B,and mTNFRSF1B) shown in (A) were inserted into the 3′UTR. Doxycycline was added to themedium to stop transcription from the tet-responsive promoter, and total cellular RNA wascollected at 0, 2, 4, and 6 hr time points. Northern blot analyses were performed to monitorGFP and β-globin (BG) mRNA levels.(C) The experiment shown in (B) was performed three times, and the northern blots werequantified using a phosphorimager. For each point, the intensity of the β-globin reporter bandwas normalized to the intensity of the GFP band, and the band intensity at the 0 hr time pointwas set at 100%. The percent of mRNA remaining was plotted over time. The error bars indicatethe standard error of the mean (SEM) from the three experiments.

Figure 2. CUGBP1 Binds Specifically to GRE Sequences with High Affinity(A) Cytoplasmic extracts from primary human T cells were mixed with a 32P-end-labeled ribo-oligonucleotide probe that contained a GRE sequence from the 3′UTR of the c-jun transcriptin the absence (No Competitor) or presence of a 100-fold molar excess of the indicatedunlabeled competitor ribo-oligonucleotides. The sequences of the GRE-containing ribo-oligonucleotides and mutated oligonucleotides are indicated as the boxed sequences in Figure1A. Bands were visualized using a phosphorimager, and the position of migration of thepredominant RNA-protein binding complex is indicated with an arrow.(B) RNA-protein gel shift assays were performed by mixing cytoplasmic extracts from primaryhuman T cells with radiolabeled ribo-oligonucleotide probes that contained GRE sequencesfrom the 3′UTR of c-jun, jun B, and TNFRSF1B or mutated GRE sequences (mc-jun, mjun B,and mTNFRSF1B) in the absence of antibody (No Antibody) or the presence of anti-CUGBP1or anti-actin antibodies. The binding reactions were then separated by electrophoresis on 10%polyacrylamide gels under nondenaturing conditions. For each panel, the position of thesupershifted band is indicated with an arrow.(C) Cytoplasmic extracts from primary human T cells (10 μg of protein) were incubated with12 fmol of a 32P end-labeled c-jun GRE probe in the absence of unlabeled RNA or the presenceof increasing amounts of unlabeled c-jun GRE RNA (12–2400 fmol in 2-fold increments). Thebinding reactions were then separated by electrophoresis on a 10% polyacrylamide gel under

non-denaturing conditions. The lane marked “P” was loaded with probe alone. The position ofmigration of the probe bound to CUGBP1 is indicated with an arrow.(D) The experiment shown in (C) was performed three times, and the amount of bound c-junGRE probe was quantified with a phosphorimager. The percent of maximal bound radiolabeledRNA was plotted against the concentration of total RNA in each reaction. Each point representsthe mean and SEM from the three experiments.(E) HeLa Tet-off cells were transfected with the pTetBBB β-globin reporter construct (NoInsert) or with reporter constructs in which the TNFRSF1B GRE sequence (TNFRSF1B) orthe mutated sequence (mTNFRSF1B) shown in Figure 1A was inserted into the 3′UTR. Lysatesfrom these transfected cells were incubated with protein G Sepharose beads that were precoatedwith anti-HA, anti-CUGBP1, or anti-PABP antibodies. RNA isolated from the input (I) andfrom the immunoprecipitation pellet (P) was assayed for the presence of β-globin (BG) orGAPDH transcripts using RT-PCR. As a control to detect possible contamination, lane 19(H2O) contained all components of the RT-PCR reaction except for an RNA sample.

Figure 3. siRNA-Mediated Knockdown of CUGBP1 Induced Stabilization of GRE-ContainingTranscriptsIn (A) and (B), HeLa Tet-off cells were treated in two rounds with two independent siRNAsdirected against CUGBP1 (siCUGBP1-A and siCGBP1-B) or a control siRNA directed againstred fluorescence protein (siControl). In (C) and (D), HeLa Tet-off cells were treated in threerounds with smart pool siRNAs directed against CUGBP1 (siCUGBP1) or nontargeting siRNA(siControl). These cells were then transfected with the pTracer GFP expression construct anda pTetBBB β-globin reporter construct in which GRE-containing sequences (TNFRSF1B orjun B) were inserted into the 3′UTR. Doxycycline was added to the medium to stoptranscription from the tet-responsive promoter, and total cellular RNA was collected at 0, 2,4, and 6 hr time points. Northern blots were performed to monitor GFP, and β-globin (BG)mRNA levels. The experiments in (A) and (C) were performed three times, the northern blotswere quantified using a phosphorimager, and the graphed results are shown in (B) and (D),respectively. For each point, the intensity of the β-globin reporter band was normalized to theintensity of the GFP band, and the band intensity at the 0 hr time point was set at 100%. Thepercent of mRNA remaining following the addition of doxycycline was plotted over time. Theerror bars indicate the standard error of the mean from the three experiments. (E) Lysates fromHeLa cells were incubated with protein G Sepharose beads that were precoated with anti-HAor anti-CUGBP1 antibodies. RNA isolated from the input (I) and from the immunoprecipitation

pellet (P) was assayed for the presence of c-jun, jun B, or GAPDH transcripts using RT-PCR.As a control to detect possible contamination, lane 5 (H2O) contained all components of theRT-PCR reaction except for an RNA sample. (F) HeLa cells were treated in two rounds withpooled siRNAs directed against CUGBP1 (siCUGBP1) or non-targeting siRNA (siControl).Actinomycin D was added to stop transcription and total cellular RNA was harvested at 0, 1,and 2 hr time points. The mRNA levels of c-jun and jun B were measured by real-time RT-PCR using transcript-specific primers, and transcript levels were normalized to the level of theHPRT transcript. The normalized level of each transcript was set at 100% at time zero, and theother time points were graphed relative to that value. Each point represents the mean and SEMfrom three independent experiments.

Table 1Examples of Short-Lived Transcripts that Contain the Consensus 11-mer GRE

Anti-CD3 + Anti-CD28-StimulatedT Cells

AffymetrixProbeSet ID Title Symbol

MedianHalf-Lifea 95% Confidence

Interval

Transcriptional Regulators

32583_at v-jun sarcoma virus 17 oncogene JUN 17 [1,22]39421_at runt-related transcription factor 1 RUNX1 18 [1,4851]40511_at GATA binding protein 3 GATA3 30 [22,36]39257_at Kruppel-like factor 12 KLF12 33 [3,4979]32087_at heat shock transcription factor 2 HSF2 56 [43,6585]

1519_atv-ets erythroblastosis virus E26 oncogenehomolog 2 ETS2 44 [18,53]

2049_s_at jun B proto-oncogene JUNB 11 [1,28]

Apoptosis Regulators

38871_at B cell CLL/lymphoma 10 BCL10 25 [3,35]

1327_s_atmitogen-activated protein kinase kinasekinase 5 MAP3K5 52 [43,85]

36463_at BCL2-associated athanogene 5 BAG5 46 [43,49]

33813_attumor necrosis factor receptor superfamily,member 1B TNFRSF1B 54 [47,69]

973_at serum/glucocorticoid-regulated kinase SGK 37 [9,54]948_s_at peptidylprolyl isomerase D (cyclophilin D) PPID 59 [43,118]

Ubiquitin Cycle and Proteolysis Regulators

41205_at ubiquitin protein ligase E3A UBE3A 52 [37,69]37662_at ubiquitin-conjugating enzyme E2G UBE2G1 59 [48,70]

Metabolism Regulators

38834_at topoisomerase (DNA) II binding protein TOPBP1 41 [32,61]

39882_attranslocase of inner mitochondrial membrane8 TIMM8A 53 [43,404]

Signal Transduction Regulators

37645_atCD69 antigen (p60, early T cell activationantigen) CD69 47 [39,56]

38311_atpapillary renal cell carcinoma (translocationassociated) TGIF2 40 [31,53]

aTranscript half-lives were measured as described in Raghavan et al. (2002).

Conserved GU-Rich Elements Mediate mRNA Decay by Binding to CUG-Binding Protein 1

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