Novel protein products encoded by upstream open reading frames of the MYCN gene in pediatric embryonal tumors.

The MYCC and MYCN loci are each associated with two upstream open reading frames (uORFs) potentially encoding small proteins (9-21 kDa). We previously demonstrated that uORFs mrtl and MYCHEX1 of MYCC are translated, and their protein products may function to regulate the expression of the "parent" oncogene. We hypothesized that a similar relationship might exist between MYCN and its two uORFs: MYCNOT and MNOP, and investigated the uORF-encoded proteins associated with MYCN to confirm their expression and intracellular location in neuroblastoma and medulloblastoma cells and tissues.

Hallmarks and Determinants of Oncogenic Translation Revealed by Ribosome Profiling in Models of Breast Cancer.

Gene expression is extensively and dynamically modulated at the level of translation. How cancer cells prioritize the translation of certain mRNAs over others from a pool of competing mRNAs remains an open question. Here, we analyze translation in cell line models of breast cancer and normal mammary tissue by ribosome profiling.

Translational control of the undifferentiated phenotype in ER‑positive breast tumor cells: Cytoplasmic localization of ERα and impact of IRES inhibition.

Using a series of potential biomarkers relevant to mechanisms of protein synthesis, we observed that estrogen receptor (ER)-positive breast tumor cells exist in two distinct yet interconvertible phenotypic states (of roughly equal proportion) which differ in the degree of differentiation and use of IRES-mediated translation. Nascently translated IGF1R in the cytoplasm positively correlated with IRES activity and the undifferentiated phenotype, while epitope accessibility of RACK1, an integral component of the 40S ribosomal subunit, aligned with the more differentiated IRES-off state. When deprived of soluble growth factors, the entire tumor cell population shifted to the undifferentiated phenotype in which IRES-mediated translation was active, facilitating survival under these adverse microenvironmental conditions.

Translational Dysregulation in Cancer: Molecular Insights and Potential Clinical Applications in Biomarker Development.

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.

IRES inhibition induces terminal differentiation and synchronized death in triple-negative breast cancer and glioblastoma cells.

Internal ribosome entry site (IRES)-mediated translation is a specialized mode of protein synthesis which malignant cells depend on to survive adverse microenvironmental conditions. Our lab recently reported the identification of a group of compounds which selectively interfere with IRES-mediated translation, completely blocking de novo IGF1R synthesis, and differentially modulating synthesis of the two c-Myc isoforms. Here, we examine the phenotypic consequences of sustained IRES inhibition in human triple-negative breast carcinoma and glioblastoma cells.

Physiological Expression and Accumulation of the Products of Two Upstream Open Reading Frames mrtl and MycHex1 Along With p64 and p67 Myc From the Human c-myc Locus.

In addition to the canonical c-Myc p64 and p67 proteins, the human c-myc locus encodes two distinct proteins, mrtl (myc-related translation/localization regulatory factor) and MycHex1 (Myc Human Exon 1), from the upstream open reading frames within the 5'-untranslated region of the c-myc P0 mRNA. The aim of this study is to examine simultaneously, for the first time, mrtl, MycHex1, c-Myc p64, and p67 in human tumor cell lines and pediatric brain tumor tissues. Western blot analysis demonstrated endogenous mrtl, MycHex1, c-Myc p64, and p67 simultaneously.

Small molecule inhibitors of IRES-mediated translation.

Many genes controlling cell proliferation and survival (those most important to cancer biology) are now known to be regulated specifically at the translational (RNA to protein) level. The internal ribosome entry site (IRES) provides a mechanism by which the translational efficiency of an individual or group of mRNAs can be regulated independently of the global controls on general protein synthesis. IRES-mediated translation has been implicated as a significant contributor to the malignant phenotype and chemoresistance, however there has been no effective means by which to interfere with this specialized mode of protein synthesis.

The human IGF1R IRES likely operates through a Shine-Dalgarno-like interaction with the G961 loop (E-site) of the 18S rRNA and is kinetically modulated by a naturally polymorphic polyU loop.

IGF1R is a proto-oncogene with potent mitogenic and antiapoptotic activities, and its expression must be tightly regulated to maintain normal cellular and tissue homeostasis. We previously demonstrated that translation of the human IGF1R mRNA is controlled by an internal ribosome entry site (IRES), and delimited the core functional IRES to a 90-nucleotide segment of the 5'-untranslated region positioned immediately upstream of the initiation codon. Here we have analyzed the sequence elements that contribute to the function of the core IRES.

Northwestern profiling of potential translation-regulatory proteins in human breast epithelial cells and malignant breast tissues: evidence for pathological activation of the IGF1R IRES.

Genes involved in the control of cell proliferation and survival (those genes most important to cancer pathogenesis) are often specifically regulated at the translational level, through RNA-protein interactions involving the 5'-untranslated region of the mRNA. IGF1R is a proto-oncogene strongly implicated in human breast cancer, promoting survival and proliferation of tumor cells, as well as metastasis and chemoresistance. Our lab has focused on the molecular mechanisms regulating IGF1R expression at the translational level.

mrtl-A translation/localization regulatory protein encoded within the human c-myc locus and distributed throughout the endoplasmic and nucleoplasmic reticular network.

mrtl (myc-related translation/localization regulatory factor) is a previously uncharacterized protein synthesized from the first open reading frame contained within the human c-myc P0 transcript, approximately 800 nucleotides upstream of the Myc coding sequence. The mrtl protein, 114 amino acids in length, is projected to contain an N-terminal transmembrane domain and a highly charged C-terminal interaction domain with homology to numerous RNA-binding proteins. Using monoclonal antibodies raised against the hydrophilic C-terminal domain, endogenous mrtl was visualized in human breast tumor cell lines and primary mammary epithelial cells at the nuclear envelope and contiguous endoplasmic/nucleoplasmic reticulum.

Alterations in RNA-binding activities of IRES-regulatory proteins as a mechanism for physiological variability and pathological dysregulation of IGF-IR translational control in human breast tumor cells.

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 ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation.

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.

Inhibition of tumorigenicity by the 5'-untranslated RNA of the human c-myc P0 transcript.

Activity of the independently regulated human c-myc P0 promoter has been associated with the undifferentiated status of leukemia cells as well as the hormone-independent proliferation of breast cancer cells. The P0 transcript is distinguished from the predominant P1 and P2 c-myc mRNAs by an approximately 639-nucleotide extension of the 5'-untranslated region. We hypothesized that this complex 5'-untranslated RNA sequence unique to the P0 transcript may contribute significantly to the composite regulation of the c-myc locus and that enforced intracellular synthesis of the isolated P0 5'-UTR, out of its native sequence context, might amplify or dominantly interfere with its normal regulatory function.

Evidence for differential ribonucleoprotein complex assembly in vitro on the 5'-untranslated region of the human IGF-IR transcript.

The type I insulin-like growth factor receptor (IGF-IR) plays a key role in the control of cellular proliferation and survival. The human IGF-IR transcript is characterized by an unusually long 1038 nucleotide 5'-untranslated region (5'-UTR). We hypothesized that the contribution of this complex 5'-untranslated RNA sequence to the post-transcriptional regulation of IGF-IR expression would involve a dynamic interplay between RNA structure and specific RNA-binding proteins.

The 5'-untranslated RNA of the human dhfr minor transcript alters transcription pre-initiation complex assembly at the major (core) promoter.

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.