GU-rich Element Research Articles

Multivalent Protein-Nucleic Acid Interactions Probed by Composition-Gradient Multiangle Light Scattering.

Many RNA-binding proteins, such as TDP-43 or CELF1, interact multivalently with nucleic acid repetitive elements. The molecular stoichiometry of protein to nucleic acid is often difficult to assess, particularly by standard electrophoretic mobility shift assays (EMSAs). Here, we investigate the use of composition-gradient multiangle light scattering (CG-MALS) for quantifying binding affinity and stoichiometry for two RNA-binding proteins with their nucleic acid partners of varied sequence and length: TDP43's N-terminal RNA recognition motifs with both TG/GU-repeat ssDNA and ssRNA, respectively, and CELF1's two N-terminal RNA recognition motifs with (TG/UGUU/GU) repeats and an experimentally defined cognate GU-rich element (GRE). Our CG-MALS data derived from each of these interactions is consistent with expected ranges of binding affinity and stoichiometry for proteins binding to shorter nucleic acid repeats. Furthermore, we conclude that CG-MALS can be an excellent method for obtaining quantitative estimates even for high (>2) protein-nucleic acid stoichiometric ratios.

Fine-tuning brassinosteroid biosynthesis via 3'UTR-dependent decay of CPD mRNA modulates wood formation in Populus.

Brassinosteroids (BRs) are plant hormones that regulate wood formation in trees. Currently, little is known about the post-transcriptional regulation of BR synthesis. Here, we show that during wood formation, fine-tuning BR synthesis requires 3'UTR-dependent decay of Populus CONSTITUTIVE PHOTOMORPHOGENIC DWARF 1 (PdCPD1). Overexpression of PdCPD1 or its 3' UTR fragment resulted in a significant increase of BR levels and inhibited secondary growth. In contrast, transgenic poplars repressing PdCPD1 3' UTR expression displayed moderate levels of BR and promoted wood formation. We show that the Populus GLYCINE-RICH RNA-BINDING PROTEIN 1 (PdGRP1) directly binds to a GU-rich element in 3' UTR of PdCPD1, leading to its mRNA decay. We thus provide a post-transcriptional mechanism underlying BRs synthesis during wood formation, which may be useful for genetic manipulation of wood biomass in trees.

CELF1 represses Doublesex1 expression via its 5' UTR in the crustacean Daphnia magna.

In sex determination of the crustacean Daphnia magna, male-specific expression of DM-domain transcription factor Doublesex1 (Dsx1) orchestrates the male developmental program triggered by environmental stimuli. We previously identified the CELF1 ortholog as a candidate of proteins associated with the 5’ UTR of the Dsx1α isoform. Here we report the CELF1-dependent suppression of Dsx1 expression in D. magna. During embryogenesis, CELF1 expression was not sexually dimorphic. Silencing of CELF1 led to the activation of Dsx1 expression both in female and male embryos. Overexpression of CELF1 in male embryos resulted in a reduction of Dsx1 expression. By these manipulations of CELF1 expression, the Dsx1 transcript level was not significantly changed. To investigate whether the CELF1 controls Dsx1 expression via its 5’ UTR, we injected the GFP reporter mRNA having intact Dsx1α 5’ UTR or mutated one lacking the GU-rich element (GRE) that is known as a binding site of the CELF1 ortholog. We found that deletion of the GRE significantly increased the reporter gene expression. These results indicate that CELF1 suppresses Dsx1 expression both in females and males, possibly at the post-transcriptional level. We speculate that CELF1 may avoid unintended Dsx1 expression and generation of sexual ambiguity by setting a threshold of Dsx1 expression.

HuR promotes miRNA-mediated upregulation of NFI-A protein expression in MDSCs during murine sepsis

Myeloid-derived suppressor cells (MDSCs) contribute to high mortality rates during sepsis, but how sepsis induces MDSCs is unclear. Previously we reported that microRNA (miR)-21 and miR-181b reprogram MDSCs in septic mice by increasing levels of DNA binding transcription factor, nuclear factor 1 (NFI-A). Here, we provide evidence that miR-21 and miR-181b stabilize NFI-A mRNA and increase NFI-A protein levels by recruiting RNA-binding proteins HuR and Ago1 to its 3′ untranslated region (3′UTR). We also find that the NFI-A GU-rich element (GRE)-binding protein CUGBP1 counters miR-21 and miR-181b dependent NFI-A mRNA stabilization and decreases protein production by replacing 3′UTR bound Ago1 with Ago2. We confirmed the miR-21 and miR-181b dependent reprogramming pathway in MDSCs transfected with a luciferase reporter construct containing an NFI-A 3′UTR fragment with point mutations in the miRNA binding sites. These results suggest that targeting NFI-A in MDSCs during sepsis may enhance resistance to uncontrolled infection.

Postsplicing-Derived Full-Length Intron Circles in the Protozoan Parasite Entamoeba histolytica.

Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5′ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5′ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5′-3′ss ligation points. Different from the reported functions of ciRNAs, the 5′ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.

TAp63γ and ΔNp63γ are regulated by RBM38 via mRNA stability and have an opposing function in growth suppression

The p63 gene is expressed as TAp63 from the P1 promoter and as ΔNp63 from the P2 promoter. Through alternative splicing, five TA and five ΔN isoforms (α-ε) are expressed. Isoforms α-β and δ share an identical 3’ untranslated region (3’UTR) whereas isoform γ has a unique 3’UTR. Recently, we found that RBM38 RNA-binding protein is a target of p63 and RBM38 in turn regulates p63α/β expression via mRNA stability. However, it is uncertain whether p63γ has a unique biological activity and whether p63γ is regulated by RBM38. Here, we found that the levels of ΔNp63γ transcript and protein are induced upon overexpression of RBM38 but decreased by RBM38 knockdown. Conversely, we found that the levels of ΔNp63β transcript and protein are decreased by ectopic expression of RBM38 but increased by RBM38 knockdown, consistent with our previous report. Interestingly, RBM38 increases the half-life of p63γ mRNA by binding to a GU-rich element in p63γ 3’UTR. In contrast, our previous studies showed that RBM38 decreases the half-life of p63α/β mRNAs by binding to AU-/U-rich elements in their 3’UTR. We also found that knockout of p63γ in ME180 and HaCaT cells, in which ΔNp63 isoforms are predominant, inhibits cell proliferation and migration, suggesting that ΔNp63γ has a pro-growth activity. In contrast, we found that knockout of TAp63γ in MIA PaCa-2 cells, in which TAp63 isoforms are predominant, promotes cell proliferation, migration, and inhibits cellular senescence. Taken together, we conclude that ΔNp63γ has an oncogenic potential whereas TAp63γ is a tumor suppressor.

Abstract A34: CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells

Abstract The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3' untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3' UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3' UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3' UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF1's regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present an eleven component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Shebna Cheema, Joseph M. Fachini, Natee Kongchan, Guojun Lu, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A34.

Abstract 3508: The RNA-binding protein CELF1 regulates the tumor suppressive protein IL-24/mda-7 expression in oral squamous cell carcinoma

Abstract The majority of oral squamous cell carcinoma (OSCC) patients are diagnosed at an advanced disease stage and the overall 5-year survival rate, surprisingly, has not changed dramatically over the past several decades. Therefore, identification and subsequent characterization of new molecular determinants of OSCC, may aid in the discovery of novel targets capable of predicting patient treatment response and disease prognosis. Co- and post-transcriptional gene expression plays a crucial role in disease development. RNA binding proteins (RBPs) are critical regulators of messenger RNA (mRNA) fate within mammalian cells. The RBP CELF1 associates with GU-rich element (GRE) containing mRNAs and post transcriptionally regulates mRNA stability, translation and pre-mRNA alternative splicing. Our previous studies demonstrated that the protein levels of CELF1, increased as HNSCC disease progressed from Stage I-IV. In addition, reduction of CELF1 in oral cancer cells promoted programmed cell death and decreased tumor burden in a xenograft mouse model. In an effort to understand the underlying mechanism employed by CELF1 to promote anti-tumor activity, we utilized genomic techniques to identify CELF1 mRNA targets in oral cancer cells. Interestingly, the gene IL-24/mda-7 was one of the top aberrantly expressed mRNAs identified as a function of CELF1 levels in oral cancer cells. Interleukin -24 also known as melanoma differentiation associated gene-7 (IL-24/mda-7) is a member of the IL-10 related family of cytokines. IL-24 protein expression is absent in most cancers and forced expression of IL-24 using an adenoviral vector enhances tumor cell-specific apoptosis. Although the mechanism of IL-24 induced cell death has been studied for several cancers, the role of IL-24 in HNSCC is not well known. In addition, the regulatory mechanisms controlling IL-24 expression in cancer are not well studied. Here, we show that IL-24 mRNA is overexpressed in HNSCC tissues compared to normal specimens. Furthermore, utilizing a mouse model of oral carcinogenesis we determined that IL-24 mRNA levels are enhanced in hyperplastic/dysplastic and oral squamous cell carcinoma tissues compared to normal, which is coincident with increased protein levels of CELF1. Surprisingly, loss of CELF1 expression in oral cancer cells dramatically increased IL-24 protein from undetectable levels to robust expression. We are currently exploring the mechanism of the CELF-1/IL-24 axis in mediating apoptosis in oral cancer cells and are expanding our studies to include other cancer types. These results suggest a novel mechanism by which the RNA binding protein CELF1 promotes oral cancer cell survival, through controlling the expression of a potent tumor suppressive protein and supports CELF1 as a viable target for the development of cancer therapeutic interventions. Citation Format: Reniqua House, Viswanathan Palanisamy. The RNA-binding protein CELF1 regulates the tumor suppressive protein IL-24/mda-7 expression in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3508.

Abstract B58: CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells

Abstract The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3′ untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3′ UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3′ UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3′ UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF1's regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present a distinctive model of gene regulation, invisible to traditional transcriptional profiling, in which the CELF1 protein serves as a central node controlling translational activation of the EMT program and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Natee Kongchan, Joseph M. Fachini, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B58.

Feedback Regulation of Kinase Signaling Pathways by AREs and GREs.

In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP.

Altered CELF1 binding to target transcripts in malignant T cells.

The RNA-binding protein, CELF1, binds to a regulatory sequence known as the GU-rich element (GRE) and controls a network of mRNA transcripts that regulate cellular activation, proliferation, and apoptosis. We performed immunoprecipitation using an anti-CELF1 antibody, followed by identification of copurified transcripts using microarrays. We found that CELF1 is bound to a distinct set of target transcripts in the H9 and Jurkat malignant T-cell lines, compared with primary human T cells. CELF1 was not phosphorylated in resting normal T cells, but in malignant T cells, phosphorylation of CELF1 correlated with its inability to bind to GRE-containing mRNAs that served as CELF1 targets in normal T cells. Lack of binding by CELF1 to these mRNAs in malignant T cells correlated with stabilization and increased expression of these transcripts. Several of these GRE-containing transcripts that encode regulators of cell growth were also stabilized and up-regulated in primary tumor cells from patients with T-cell acute lymphoblastic leukemia. Interestingly, transcripts encoding numerous suppressors of cell proliferation that served as targets of CELF1 in malignant T cells, but not normal T cells, exhibited accelerated degradation and reduced expression in malignant compared with normal T cells, consistent with the known function of CELF1 to mediate degradation of bound transcripts. Overall, CELF1 dysfunction in malignant T cells led to the up-regulation of a subset of GRE-containing transcripts that promote cell growth and down-regulation of another subset that suppress cell growth, producing a net effect that would drive a malignant phenotype.

In silico characterization of a RNA binding protein of cattle filarial parasite Setaria digitata.

Human lymphatic filariasis (HLF) is a neglected tropical disease which threatens nearly 1.4 billion people in 73 countries worldwide. Wuchereria bancrofti is the major causative agent of HLF and it closely resembles cattle filarial parasite Setaria digitata. Due to difficulties in procuring W. bancrofti parasite material, S. digitata cDNA library has been constructed to identify novel drug targets against HLF and many of the cDNA sequences are yet to be assigned structure and function. In this study, a 549 bp long cDNA (sdrbp) has been sequenced and characterized in silico. The shortest ORF of 249 bp from the isolated cDNA encodes a polypeptide of 82 amino acids and shows an amino acid identity of 54% with the RRM domain of human cleavage stimulation factor-64 kDa subunit (CstF-64). Structure of the protein (sdRBP) obtained by homology modelling using RRM of CstF-64 as template adopts classical RRM topology (β1α1β2β3α2β4). sdRBP model built was validated by superimposition tools and Ramachandran plot analysis. CstF-64 plays an important role in pre-mRNA polyadenylation by interacting with specific GU-rich downstream sequence element. Molecular docking studies of sdRBP with different RNA molecules revealed that sdRBP has greater binding affinity to GU-rich RNA and comparable results were obtained upon similar docking of RRM of CstF-64 with the same RNA molecules. Therefore, sdRBP is likely to perform homologous function in S. digitata. This study brings new dimensions to the functional analysis of RNA binding proteins of S. digitata and their evaluation as new drug targets against HLF.

Isolation and characterization of rl (t), a gene that controls leaf rolling in rice

Moderate leaf rolling is one of the most important morphological traits in rice breeding for plant ideotype. Previous studies have shown that the rl (t) gene has a high breeding potential for developing hybrid-rice varieties with an ideal ideotype, because it leads to an appropriate leaf rolling index (LRI) of about 30 % in the heterozygous state, and had a positive effect on grain yield. In this study, we isolated rl (t) and performed a preliminary investigation of its function in regulating leaf rolling in rice. DNA sequencing identified a single base change (G to T) in the finely mapped region (11 kb) containing rl (t), and this is located in 3′-untranslated region (3′-UTR) of the only predicted gene, Roc5 (Rice outermost cell-specific). The expression level of Roc5 is significantly higher in the rl (t) mutant than in the wild-type. Using RNAi and overexpression analysis, we found that the expression level of Roc5 correlated with LRI and leaf bulliform area, and was also associated with leaf abaxial or adaxial rolling. These results confirmed that Roc5 controls leaf rolling in a dosage-dependent manner. Bioinformatics analysis revealed a conserved 17-nt sequence (called the GU-rich element) in the 3′-UTR of HD-GL2 (Homeodomain-Glabra2) family genes including Roc5. Based on the model of this element in regulating mRNA stability in mammals, we speculate that the single nucleotide change in this element accounts for the higher expression level of Roc5 in the rl (t) mutant compared to the wild-type, which ultimately leads to adaxial rolling of the leaf. This discovery further enhances our knowledge of the molecular mechanisms underlying leaf rolling in rice.

Circular Intronic Long Noncoding RNAs

We describe the identification and characterization of circular intronic long noncoding RNAs in human cells, which accumulate owing to a failure in debranching. The formation of such circular intronic RNAs (ciRNAs) can be recapitulated using expression vectors, and their processing depends on a consensus motif containing a 7 nt GU-rich element near the 5' splice site and an 11 nt C-rich element close to the branchpoint site. In addition, we show that ciRNAs are abundant in the nucleus and have little enrichment for microRNA target sites. Importantly, knockdown of ciRNAs led to the reduced expression of their parent genes. One abundant such RNA, ci-ankrd52, largely accumulates to its sites of transcription, associates with elongation Pol II machinery, and acts as a positive regulator of Pol II transcription. This study thus suggests a cis-regulatory role of noncoding intronic transcripts on their parent coding genes.

Overexpression of RNA-binding protein CELF1 prevents apoptosis and destabilizes pro-apoptotic mRNAs in oral cancer cells

CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA’s encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3′UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3′UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.

Coordinate regulation of mRNA decay networks by GU-rich elements and CELF1

The GU-rich element (GRE) was identified as a conserved sequence enriched in the 3' UTR of human transcripts that exhibited rapid mRNA turnover. In mammalian cells, binding to GREs by the protein CELF1 coordinates mRNA decay of networks of transcripts involved in cell growth, migration, and apoptosis. Depending on the context, GREs and CELF1 also regulate pre-mRNA splicing and translation. GREs are highly conserved throughout evolution and play important roles in the development of organisms ranging from worms to man. In humans, abnormal GRE-mediated regulation contributes to disease states and cancer. Thus, GREs and CELF proteins serve critical functions in gene expression regulation and define an important evolutionarily conserved posttranscriptional regulatory network.

Polyadenylation: alternative lifestyles of the A-rich (and famous?)

Polyadenylation: alternative lifestyles of the A-rich (and famous?)

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

We 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 consensus sequence UGUUUGUUUGU, which we have termed a GU-rich element (GRE), was enriched in short-lived transcripts. Using a tet-off reporter system, we showed that insertion of GRE-containing sequences from c-jun, jun B, or TNF receptor 1B, but not mutated GRE sequences, into the 3'UTR of a beta-globin transcript conferred instability on the otherwise stable beta-globin transcript. CUG-binding protein 1 (CUGBP1) was identified as the major GRE-binding activity in cytoplasmic extracts from primary human T cells based on supershift and immunoprecipitation assays. siRNA-mediated knockdown of CUGBP1 in HeLa cells caused stabilization of GRE-containing transcripts, suggesting that CUGBP1 is a mediator of GRE-dependent mRNA decay. Overall, our results suggest that the GRE mediates coordinated mRNA decay by binding to CUGBP1.

GU-Rich RNA: Expanding CUGBP1 Function, Broadening mRNA Turnover

In this issue of Molecular Cell, Vlasova et al. (2008) identify the GU-rich element (GRE) as a novel, widespread, degradation-promoting sequence through which the RNA-binding protein CUGBP1 elicits mRNA decay.