The promoter of the human dihydrofolate reductase (DHFR)

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5′-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.

The c-myc protooncogene plays an important role in the regulation of cellular proliferation. Mithramycin, a DNA binding antibiotic which binds G-C-rich DNA, inhibits c-myc expression in both differentiating and nondifferentiating cells. The G-C-rich nature of the c-myc promoter suggests that mithramycin may act by directly inhibiting promoter function. The mithramycin binding sites in the c-myc promoter regions were determined by DNAse I footprinting. Particularly prominent mithramycin binding is noted in the regions just 5' of the P1 and P2 promoter TATA boxes. Gel retardation experiments performed in the presence of mithramycin demonstrate that drug binding can prevent the formation of discrete complexes between HeLa cell nuclear proteins and c-myc promoter DNA fragments. Mithramycin also directly blocks the binding of the transcription factor Sp1 to the P1 promoter region. In vitro run-off transcription demonstrates that mithramycin can completely inhibit the in vitro function of both the P1 and P2 promoters. These data suggest that mithramycin inhibits transcription of the c-myc protooncogene by blocking the binding of important regulatory factors, thus preventing formation of the c-myc transcription initiation complex.

The type I insulin-like growth factor receptor (IGF-IR) is integrally involved in the control of cellular proliferation and survival. An internal ribosomal entry site (IRES) within the 1,038 nucleotide 5'-untranslated region of the human IGF-IR mRNA helps to provide the tight control of IGF-IR expression necessary for maintenance of normal cellular and tissue homeostasis. The IRES maps to a discrete sequence of 85 nucleotides positioned just upstream of the IGF-IR initiation codon, allowing the ribosome to bypass the highly structured regions of the 5'-UTR as well as the upstream open reading frame. The authenticity of the IGF-IR IRES has been confirmed by its sensitivity to deletion of the promoter from a bicistronic reporter construct, and its resistance in a monocistronic reporter construct to co-expression of a viral 2A protease. We previously characterized HuR as a potent repressor of IGF-IR translation. Here we demonstrate that hnRNP C competes with HuR for binding the IGF-IR 5'-UTR and enhances IRES-mediated translation initiation in a concentration-dependent manner. We observed changes in binding of hnRNP C versus HuR to the IGF-IR 5'-UTR in response to physiological alterations in cellular environment or proliferative status. Furthermore, we have found distinct alterations in the pattern of protein binding to the IGF-IR 5'-UTR in human breast tumor cells in which IGF-IR IRES activity and relative translational efficiency are aberrantly increased. These results suggest that dysregulation of the IGF-IR IRES through changes in the activities of RNA-binding translation-regulatory proteins could be responsible for IGF-IR overexpression in a proportion of human breast tumors.

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.

Although transcript levels have been traditionally used as a surrogate measure of gene expression, it is increasingly recognized that the latter is extensively and dynamically modulated at the level of translation (messenger RNA to protein). Over the recent years, significant progress has been made in dissecting the complex posttranscriptional mechanisms that regulate gene expression. This advancement in knowledge came hand in hand with the progress made in the methodologies to study translation both at gene-specific as well as global genomic level. The majority of translational control is exerted at the level of initiation; nonetheless, protein synthesis can be modulated at the level of translation elongation, termination, and recycling. Sequence and structural elements and epitranscriptomic modifications of individual transcripts allow for dynamic gene-specific modulation of translation. Cancer cells usurp the regulatory mechanisms that govern translation to carry out translational programs that lead to the phenotypic hallmarks of cancer. Translation is a critical nexus in neoplastic transformation. Multiple oncogenes and signaling pathways that are activated, upregulated, or mutated in cancer converge on translation and their transformative impact “bottlenecks” at the level of translation. Moreover, this translational dysregulation allows cancer cells to adapt to a diverse array of stresses associated with a hostile microenviroment and antitumor therapies. All elements involved in the process of translation, from the transcriptional template, the components of the translational machinery, to the proteins that interact with the transcriptome, have been found to be qualitatively and/or quantitatively perturbed in cancer. This review discusses the regulatory mechanisms that govern translation in normal cells and how translation becomes dysregulated in cancer leading to the phenotypic hallmarks of malignancy. We also discuss how dysregulated mediators or components of translation can be utilized as biomarkers with potential diagnostic, prognostic, or predictive significance. Such biomarkers have the potential advantage of uniform applicability in the face of inherent tumor heterogeneity and deoxyribonucleic acid instability. As translation becomes increasingly recognized as a process gone awry in cancer and agents are developed to target it, the utility and significance of these potential biomarkers is expected to increase.

The human dhfr minor transcript is distinguished from the predominant dhfr mRNA by an approximately 400 nucleotide extension of the 5'-untranslated region, which corresponds to the major (core) promoter DNA (its template). Based on its unusual sequence composition, we hypothesized that the minor transcript 5'-UTR might be capable of altering transcription pre-initiation complex assembly at the core promoter, through direct interactions of the RNA with specific regulatory polypeptides or the promoter DNA itself. We found that the minor transcript 5'-UTR selectively sequesters transcription factor Sp3, and to a lesser extent Sp1, preventing their binding to the dhfr core promoter. This allows a third putative transcriptional regulatory protein, which is relatively resistant to sequestration by the minor transcript RNA, the opportunity to bind the dhfr core promoter. The selective sequestration of Sp3 > Sp1 by the minor transcript 5'-UTR involves an altered conformation of the RNA, and a structural domain of the protein distinct from that required for binding to DNA. As a consequence, the minor transcript 5'-UTR inhibits transcription from the core promoter in vitro (in trans) in a concentration-dependent manner. These results suggest that the dhfr minor transcript may function in vivo (in cis) to regulate the transcriptional activity of the major (core) promoter.

The ability of oligodeoxynucleotides to form specific triple helical structures with critical regulatory sequences in the human dihydrofolate reductase (DHFR) promoter was investigated. A battery of purine-rich oligonucleotides targeted to the two purine.pyrimidine strand biased regions near the DHFR transcription initiation site was developed. The stable triple helical structures formed by binding of the oligonucleotides to the native promoter double helix were dominated by G*G.C triplets, with interspersed C*C.G and A*A.T alignments. Mismatches between the oligonucleotide and the purine-rich strand of the target significantly destabilized third strand binding, and a G*A.T alignment was particularly unfavorable. Formation of a pur.pur.pyr triple helical structure results in a localized limitation of access to the native double helical DNA and produces sequence dependent conformational alterations extending several nucleotides beyond the triplex-duplex boundary. Although they differ only by the insertion of two A.T base pairs, the distal and proximal purine.pyrimidine regions can be targeted individually due to the high degree of sequence specificity of triple helical alignment. Triplex formation overlapping any of three consensus transcriptional regulatory elements and collectively covering 50% of the DHFR core promoter is now possible with this set of oligonucleotides.