Transcriptional regulation and functional analysis of the human Wilms' tumor 1 gene

The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription factor and is expressed in urogenital, hematopoietic and other tissues. It is expressed in a temporal and spatial manner in both embryonic and adult stages. To obtain a better understanding of the biological function of WT1, we studied two aspects of WT1 regulation: one is the identification of tissue-specific cis-regulatory elements that regulate its expression, the other is the downstream genes which are modulated by WT1.^ My studies indicate that in addition to the promoter, other regulatory elements are required for the tissue specific expression of this gene. A 259-bp hematopoietic specific enhancer in intron 3 of the WT1 gene increased the transcriptional activity of the WT1 promoter by 8- to 10-fold in K562 and HL60 cells. Sequence analysis revealed both GATA and c-Myb motifs in the enhancer fragment. Mutation of the GATA motif decreased the enhancer activity by 60% in K562 cells. Electrophoretic mobility shift assays showed that both GATA-1 and GATA-2 proteins in K562 nuclear extracts bind to this motif. Cotransfection of the enhancer containing reporter construct with a GATA-1 or GATA-2 expression vector showed that both GATA-1 and GATA-2 transactivated this enhancer, increasing the CAT reporter activity 10-15 fold and 5-fold respectively. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT reporter construct and a c-Myb expression vector showed that c-Myb transactivated the enhancer by 5-fold. A DNase I-hypersensitive site has been identified in the 258 bp enhancer region. These data suggest that GATA-1 and c-Myb are responsible for the activity of this enhancer in hematopoietic cells and may bind to the enhancer in vivo. In the process of searching for cis-regulatory elements in transgenic mice, we have identified a 1.0 kb fragment that is 50 kb downstream from the promoter and is required for the central nervous system expression of WT1.^ In the search for downstream target genes of WT1, we noted that the proto-oncogene N-myc is coexpressed with the tumor suppressor gene WT1 in the developing kidney and is overexpressed in many Wilms' tumors. Sequence analysis revealed eleven consensus WT1 binding sites located in the 1 kb mouse N-myc promoter. We further showed that the N-myc promoter was down-regulated by WT1 in transient transfection assays. Electrophoretic mobility shift assays showed that oligonucleotides containing the WT1 motifs could bind WT1 protein. Furthermore, a Denys-Drash syndrome mutant of WT1, R394W, that has a mutation in the DNA binding domain, failed to repress the N-myc promoter. This suggests that the repression of the N-myc promoter is mediated by DNA binding of WT1. This finding helps to elucidate the relationship of WT1 and N-myc in tumorigenesis and renal development. ^

Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^

A urokinase receptor promoter element (-152/-135) bound with an AP-2-alpha-related protein and SP1 is required for the induction of gene expression by PMA and SRC

The urokinase-type plasminogen activator receptor (u-PAR) promotes extracellular matrix degradation, invasion and metastasis. A first objective of this dissertation was to identify cis-elements and trans-acting factors activating u-PAR gene expression through a previously footprinted (–148/–124) promoter region. Mobility shifting experiments on nuclear extracts of a high u-PAR-expressing colon cancer cell line (RKO) indicated Sp1, Sp3 and a factor similar to, but distinct from, AP-2α bound to an oligonucleotide spanning –152/–135. Mutations preventing the binding of the AP-2α-related factor reduced u-PAR promoter activity. In RKO, the expression of a dominant negative AP-2 (AP-2αB) diminished u-PAR promoter activity, protein and u-PAR mediated laminin degradation. Conversely, u-PAR promoter activity in low u-PAR-expressing GEO cells was increased by AP-2αA expression. PMA treatment, which induces u-PAR expression, caused an increased amount of the AP-2α-related factor-containing complex in GEO, and mutations preventing AP-2α-like and Sp1/Sp3 binding reduced the u-PAR promoter stimulation by PMA. In resected colon cancers, u-PAR protein amounts were related to the amount of the AP-2α-related factor-containing complex. In conclusion, constitutive and PMA- inducible u-PAR gene expression and -proteolysis are mediated partly through transactivation via a promoter sequence (–152/435) bound with an AP-2α-related factor and Sp1/Sp3. ^ A second interest of this dissertation was to determine if a constitutively active Src regulates the transcription of the u-PAR gene, since c-src expression increases invasion in colon cancer. Increased u-PAR protein and laminin degradation paralleling elevated Src activity was evident in SW480 colon cancer cells stably expressing a constitutively active Src (Y- c-src527F). Nuclear run-on experiments indicated that this was due largely to transcriptional activation. While transient transfection of SW480 cells with Y-c-src527F induced a u-PAR-CAT-reporter, mutations preventing Sp1-binding to promoter region –152/435 abolished this induction. Mobility shift assays revealed increased Sp1 binding to region –152/135 with nuclear extracts of Src-transfected SW480 cells. Finally, the amounts of endogenous u-PAR in resected colon cancers significantly correlated with Src-activity. These data suggest that u-PAR gene expression and proteolysis are regulated by Src, this requiring the promoter region (–152/–135) bound with Sp1, thus, demonstrating for the first time that transcription factor Sp1 is a downstream effector of Src. ^

Transcriptional regulation of the human Fas (CD95) promoter

The coordination of the apoptotic program necessitates the timely expression of sensor, effector, and mediator molecules. Fas/CD95, a transmembrane receptor which tethers the cell-death machinery, triggers apoptosis to maintain immune homeostasis, tolerance, and surveillance. Dysregulation in Fas-mediated apoptosis, either from disproportionate expression or disruptions in the downstream signaling pathway, manifests in autoimmune disorders and certain malignant progression. ^ In this project, the transcriptional requirements underlying two modulators of Fas expression were investigated. In T-lymphocytes, activation results in potent Fas upregulation followed by an acquisition of sensitivity towards FasL-mediated apoptosis. Human fas promoter cloning and analysis have identified a cis-element critical for inducible Fas expression. EMSA studies using this region demonstrated a constitutive association with the transcription factor Sp1 and inducible NF-κB binding in response to activation. These interactions were mutually exclusive, as the rB/Sp1 element bound with recombinant Sp1 was readily displaced by increasing amounts of NF-κB p50. Thus, Fas upregulation by T-cell activation stimuli is dependent upon NF-κB binding at the fas promoter. ^ The capacity of Sp1 to direct basal Fas expression was examined through mutagenesis of several GC-rich regions within the core fas promoter. Reporter analysis of single or combinatorial mutant GC-box constructs revealed usage of a particular GC-element in moderating over 50% of basal fas transcription. Inducible expression was Sp1-independent, however, since activated Jurkat cells containing fas Sp1-mutant constructs retained equivalent reporter induction. Overall, a dual-level of transcriptional control exists in fas, where constitutive activity is monitored through Sp1 binding, whereas T-cell activation obligates NF κB transactivation. ^ In response to genotoxic damage, p53 modulates Fas levels partly by a transcription-dependent mechanism. Reconstitution of wild-type p53 in the hepatoma cell line Hep3B readily induced Fas transcription. Furthermore, fas promoter analysis identified an undescribed p53 responsive element which, when deleted, ablated p53-mediated reporter activity. Therefore, the pro-apoptotic function mediated by p53 is driven partially through the enhancement of Fas expression. ^ Altogether, events elicting Fas transcription may invoke single or overlapping mechanisms that converge at the level of promoter activity. Agents that enhance or attenuate these pathways may be therapeutically beneficial in modulating the expression and sensitivity towards Fas-dependent apoptosis. ^

Estrogen action during early mouse mammary gland development

Estrogens have been implicated in the normal and neoplastic development of the mammary gland. Although estradiol is essential for early mammary differentiation, its role in postnatal ductal morphogenesis is poorly defined. We have found that neonatal estradiol exposure promotes precocious ductal outgrowth and terminal end bud formation in 21 day-old female mice. In contrast to this precocious phenotype, day 21 estradiol-treated epithelium, transplanted into control host fatpads, grows more slowly than control epithelium. Western and immunohistochemical (IHC) analyses indicate that neonatally-estrogenized glands have significantly less total ER than controls at days 7 and 21, and significantly more stromal ER at day 35. Estrogen receptor α (ER) is present in the gland when treatment is initiated at day 1. We propose that the premature activation of ER by neonatal estradiol exposure, during this critical perinatal period, is a key factor in the alteration of mammary growth and ER expression. ^ To address the role of ER function in mammary morphogenesis, we have developed an in vitro system to study the effect of estradiol exposure in vivo. Keratin and ER-positive mammary epithelial cell lines from 7, 21 and 35 day-old oil or estradiol treated mice have been established. Cell lines derived from estradiol-treated mice grow significantly slower than cells from control glands. Although the level of ER expressed by each cell line is correlated to its rate of growth, epithelial growth in vitro is estradiol-independent and antiestrogen-insensitive. Estradiol-induced transcription from an ERE-reporter in transiently-transfected cell lines confirms the functionality of the ER detected by western and IHC. However, there are no differences in estradiol-stimulated transcription between cell lines. ^ In conclusion, neonatal estradiol treatment alters the pattern of ER expression in mammary epithelial and stromal cells in vivo, and the growth of mammary epithelial cells in vivo and in vitro. When grown outside of the estrogenized host, exposed epithelium grows more slowly than the control. Therefore, an extra-epithelial factor is necessary for enhanced epithelial growth. Our model, which couples an in vivo-in vitro approach, can be used in the future to identify factors involved in the period of early mammary outgrowth and carcinogen susceptibility. ^

Transcription factor SEF: Purification and functional characterization

Cytokine-induced transcription of the serum amyloid A3 (SAA3) gene promoter requires a transcriptional enhancer that contains three functional elements: two C/EBP-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa cell nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA-affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing, oligonucleotide competition and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression plasmid with SAA3-luciferase reporter resulted in 3- to 5-fold activation of SAA3 promoter. Interestingly, when SEF-transfected cells were treated with either conditioned medium (CM) or interleukin (IL) 1, the SAA3 promoter was synergistically activated in a dose-dependent manner. Furthermore, when SEF-binding site was mutated, the response of SAA3 promoter to IL-1 or CM stimulation was abolished or drastically decreased, suggesting that SEF may functionally cooperate with an IL-1-inducible transcription factor. Indeed, our functional studies showed that NFκB is a key transcription factor that mediates the IL-1-induced expression of SAA3 gene, and that SEF can synergize with NFκBp65 to activate SAA3 promoter. By coimmunoprecipitation experiments, we found that SEF could specifically interact with NFκBp65, and that the association of these two factors was enhanced upon IL-1 and CM stimulation. This suggests that the molecular basis for the functional synergy between SEF and NFκB may be due to the ability of SEF to physically interact with NPκB. In addition to its interaction with SEF, NFκB-dependent activation also requires the weak κB site in the C element and its interaction with C/EBP. Besides its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of α2-macroglobulin, Aα fibrinogen, and 6–16 genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. By coimmunoprecipitation experiments, we determined that SEF could specifically associate with both Stat3 and Stat2 upon cytokine stimulation. To examine the functional roles of such interactions, we evaluated the effects of SEF on the transcriptional regulation of two reporter genes: Aα fibrinogen and 6–16, which are IL-6- and interferon-α-responsive, respectively. Our results showed that cotransfection of SEF expression plasmid can activate the expression of Aα fibrinogen gene and 6–16 gene. Moreover, SEF can dramatically enhance the interferon-α-induced expression of 6–16 gene and IL-6-induced expression of Aα fibrinogen gene, suggesting that SEF may functionally cooperate with ISGF3 and Stat3 to mediate interferon-α and IL-6 signaling. ^ Our findings that SEF can interact with multiple cytokine-inducible transcription factors to mediate the expression of target genes open a new avenue of investigation of cooperative transcriptional regulation of gene expression, and should further our understanding of differential gene expression in response to a specific stimulus. In summary, our data provide evidence that SEF can mediate the signaling of different cytokines by interacting with various cytokine-inducible transcription factors. ^

UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells

UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.

Cytochrome P450 Regulation by -Tocopherol in Pxr-Null and PXR-Humanized Mice

The pregnane X receptor (PXR) has been postulated to play a role in the metabolism of α-tocopherol owing to the up-regulation of hepatic cytochrome P450 (P450) 3A in human cell lines and murine models after α-tocopherol treatment. However, in vivo studies confirming the role of PXR in α-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized (hPXR), wild-type, and Pxr-null mouse models were used to determine whether α-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major α-tocopherol metabolites was observed among the hPXR, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for α-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by α-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, hPXR, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that α-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by α-tocopherol. The implications of a novel role for α-tocopherol in Cyp2c gene regulation are also discussed.

A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities.

Mechanistic aspects of NMD in human cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with 3′ UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of translation. However, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Mechanistic aspects of mRNA targeting for nonsense-mediated mRNA decay in human cells

The nonsense-mediated mRNA decay (NMD) pathway is best known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with ORF-truncating premature termination codons (PTCs), but a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD substrates. We try to decipher the mechanism of mRNA targeting to the NMD pathway in human cells. Recruitment of the conserved RNA-binding helicase UPF1 to target mRNAs has been reported to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3’ untranslated region (UTR) of mRNAs has also been reported. We have transcriptome-wide determined the UPF1 binding sites by individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation. We detected a strongly enriched association of UPF1 with 3’ UTRs in undisturbed, translationally active cells. After translation inhibition, a significant increase in UPF1 binding to coding sequence (CDS) was observed, indicating that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This suggests that the decision to trigger NMD occurs after association of UPF1 with mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. In a second recent study, we re-visited the reported restriction of NMD in mammals to the ‘pioneer round of translation’, i.e. to cap-binding complex (CBC)-bound mRNAs. The limitation of mammalian NMD to early rounds of translation would indicate a – from an evolutionary perspective – unexpected mechanistic difference to NMD in yeast and plants, where PTC-containing mRNAs seem to be available to NMD at each round of translation. In contrast to previous reports, our comparison of decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells revealed that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E‐bound mRNPs are substrates for nonsense‐mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E‐bound mRNPs are substrates for nonsense‐mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.