Putative Internal Ribosome Entry Site Research Articles

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5′-untranslated region (5′ UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

Identification of an IRES within the coding region of the structural protein of human rhinovirus 16.

As an alternative mechanism for cap-dependent (m7GpppN) translation, internal ribosome entry site (IRES)-dependent translation has been observed in the 5' untranslated regions (5' UTR) and coding regions of a number of viral and eukaryotic mRNAs. In this study, a series of 5' terminal truncated structural protein genes that were fused with GFP was used to screen for potential IRESs, and IRESs were identified using a bicistronic luciferase vector or GFP expression vector possessing a hairpin structure. Our results revealed that a putative IRES was located between nt 1982 and 2281 in the VP3 coding region of the human rhinovirus 16 (HRV16) genomes. We also demonstrated that effective IRES-initiated protein expression in vitro did not occur through splicing sites or cryptic promoters. We confirmed that thapsigargin (TG), an inducer of endoplasmic reticulum stress (ERS), facilitated increased IRES activity in a dose-dependent manner. Additionally, the secondary structure of the IRES was predicted online using the RNAfold web server.

Identification of cryptic putative IRESs within the ORF encoding the nonstructural proteins of the human rhinovirus 16 genome

Internal ribosome entry site (IRES)-dependent translation is a mechanism distinct from 5' cap-dependent translation. IRES elements are located mainly in the 5' untranslated regions (UTRs) of viral and eukaryotic mRNAs. However, IRESs are also found in the coding regions of some viral and eukaryotic genomes to initiate the translation of some functional truncated isoforms. Here, five putative IRES elements of human rhinovirus 16 (HRV16) were identified in the coding region of the nonstructural proteins P2 and P3 through fusion with green fluorescent protein (GFP) expression vectors and bicistronic vectors with a hairpin structure. These five putative IRESs were located at nucleotide positions 4286-4585, 5002-5126, 6245-6394, 6619-6718, and 6629-6778 in the HRV16 genome. The functionality of the five IRESs was confirmed by their ability to initiate GFP expression in vitro. This suggests that an alternative mechanism might be used to increase the efficiency of replication of HRV16.

Human IRES Atlas: an integrative platform for studying IRES-driven translational regulation in humans

It is now known that cap-independent translation initiation facilitated by internal ribosome entry sites (IRESs) is vital in selective cellular protein synthesis under stress and different physiological conditions. However, three problems make it hard to understand transcriptome-wide cellular IRES-mediated translation initiation mechanisms: (i) complex interplay between IRESs and other translation initiation–related information, (ii) reliability issue of in silico cellular IRES investigation and (iii) labor-intensive in vivo IRES identification. In this research, we constructed the Human IRES Atlas database for a comprehensive understanding of cellular IRESs in humans. First, currently available and suitable IRES prediction tools (IRESfinder, PatSearch and IRESpy) were used to obtain transcriptome-wide human IRESs. Then, we collected eight genres of translation initiation–related features to help study the potential molecular mechanisms of each of the putative IRESs. Three functional tests (conservation, structural RNA–protein scores and conditional translation efficiency) were devised to evaluate the functionality of the identified putative IRESs. Moreover, an easy-to-use interface and an IRES–translation initiation interaction map for each gene transcript were implemented to help understand the interactions between IRESs and translation initiation–related features. Researchers can easily search/browse an IRES of interest using the web interface and deduce testable mechanism hypotheses of human IRES-driven translation initiation based on the integrated results. In summary, Human IRES Atlas integrates putative IRES elements and translation initiation–related experiments for better usage of these data and deduction of mechanism hypotheses. Database URL: http://cobishss0.im.nuk.edu.tw/Human_IRES_Atlas/

Abstract A65: Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform

Abstract Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also known as 611CTF) is a hyperactive truncated ERBB2 isoform capable of increasing cell migration and invasion in multiple cell types in vitro, and induction of human breast epithelial cell (HMLE) xenograft formation in vivo (Ward et al., Oncogene 2013;32(19):2463-74). p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, though the mechanism to account for such cap-independent p110 t-ERBB2 translation is not well studied. mRNA structural elements termed internal ribosome entry sites (IRESs) can initiate translation in a cap-independent manner when canonical cap-dependent translation is severely compromised. It has been previously demonstrated that the human EGFR 5’ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an IRES (Webb TE, et al., Oncogenesis 2015;4:e134). Therefore, we sought to identify presence of a putative IRES within the 5′ UTR that might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis. Methods: The ERBB2 mRNA 5 ′ UTR from 2 published human ERBB2 transcripts, and several overlapping sequences from full-length ERBB2 start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and promoter-less vector pRFΔP (SV40 promoter removed by restriction digestion), then the resultant constructs were transiently transfected into different cell lines (BT474, SK-BR3, MCF-7, HeLa, CHO). Three control constructs pRF (empty vector control), pRF-Tub (negative control, containing β-tubulin 5 ′ UTR, which lacks IRES activity), and pRF-myc (positive control, containing the well-characterized c-myc IRES) were parallel-transfected. Luciferase expression was then quantified using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer’s instructions. Parallel Western blot analysis and qRT-PCR were also conducted. Results: In this report, we demonstrate that in BT474 and SK-BR3 cells, no IRES activity was detected within human ERBB2 5 ′ UTR sequences under nonstressed conditions, or under serum-starvation, hypoxic conditions, or thapsgargin-induced endoplasmic reticulum stress—conditions when global translation was compromised. The construct pRF-+265/+1561 (within ERBB2 mRNA coding sequence, 5 ′ to the p110 t-ERBB2 start codon) displayed a 10-21 fold increase in firefly/Renilla activity when compared with the empty control pRF and negative control pRF-Tub, consistent with the possibility that the region between +265/+1561 may contain a cryptic promoter. Conclusions: These data are inconsistent with the hypothesis that a 5 ′ UTR IRES-mediated mechanism is involved in the translation of p110 t-ERBB2 isoform, and that other mechanisms are operative in alternative translational regulation/biosynthesis of p110 t-ERBB2 isoform. Citation Format: Yu Zong, Yulin Li, Xiaofei Liu, Mark Daniel Pegram. Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A65.

Abstract P6-05-05: Discistronic reporter screen for internal ribosome entry site (IRES) - mediated translational regulation of truncated p110 ERBB2 isoform

Abstract Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also as 611CTF) is a hyperactive truncated ERBB2 isoform capable of increasing cell migration and invasion in multiple cell types in vitro, and induction of human breast epithelial cell (HMLE) xenograft formation in vivo [Ward, et al., Oncogene (2013) 32, 2463–2474]. p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, however, it is unclear how regulation of its expression may be achieved. mRNA structural elements termed internal ribosome entry sites (IRESs) can initiate translation in a cap-independent manner when canonical cap-dependent translation is severely compromised. By cloning the EGFR 5' untranslated region (UTR) between the Renilla and firefly luciferase open reading frames of pRF, Webb, et al. have reported human EGFR 5' UTR sequence can initiate expression of a downstream open reading frame via an IRES [Oncogenesis (2014) 3, e134]. Therefore, we sought to identify presence of a putative IRES within the 5'UTR ERBB2 mRNA which might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis. Methods: Discistronic reporter pRF was used as the backbone vector to detect IRES activity. Promoter-less vector pRFΔP were constructed by removing SV40 promoter via restriction digestion. The HER2 mRNA 5'UTR (from both variant 1 and variant 3) and several overlapping sequences from full length ERBB2 (p185 ERBB2) start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and pRFΔP, then the resultant constructswere transiently transfected into different cell lines(BT474, SK-BR3, MCF-7, HeLa, CHO). Three control constructs pRF (empty vector control), pRF-Tub (negative control, containing βtubulin 5'UTR, which lacks IRES activity) and pRF-myc (positive control, containing the well-characterized c-myc IRES) were parallel-transfected. Luciferase expression was then quantified using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer's instructions. Parallel western blot analysis and qRT-PCR were also conducted. Results: In this report, we demonstrate that in BT474 and SK-BR3 cells, no IRES activity was detected within human ERBB2 5'UTR sequences under non-stressed conditions, or under serum-starvation, hypoxic conditions or thapsgargin-induced endoplasmic reticulum stress -- conditions when global translation was compromised.The construct pRF-+265/+1561 (within ERBB2 mRNA coding sequence, 5' to the p110 t-ERBB2 start codon) displayed a 10-21 fold increase in firefly/Renilla activity when compared with the empty control pRF and negative control pRF-Tub,consistent with the possibility that the region between +265/+1561 may contain a cryptic promoter. Conclusions: These data are inconsistent with the hypothesis that a 5'UTR IRES-mediated mechanism is involved in the translation of p110 t-ERBB2 isoform, and that other mechanisms are operative in alternative translational regulation/biosynthesis of p110 ERBB2 isoform. Citation Format: Zong Y, Li Y, Liu X, Pegram MD. Discistronic reporter screen for internal ribosome entry site (IRES) - mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-05.

Identification of new TSGA10 transcript variants in human testis with conserved regulatory RNA elements in 5'untranslated region and distinct expression in breast cancer

Testis specific gene antigen 10 (TSGA10) is a cancer testis antigen involved in the process of spermatogenesis. TSGA10 could also play an important role in the inhibition of angiogenesis by preventing nuclear localization of HIF-1α. Although it has been shown that TSGA10 messenger RNA (mRNA) is mainly expressed in testis and some tumors, the transcription pattern and regulatory mechanisms of this gene remain largely unknown. Here, we report that human TSGA10 comprises at least 22 exons and generates four different transcript variants. It was identified that using two distinct promoters and splicing of exons 4 and 7 produced these transcript variants, which have the same coding sequence, but the sequence of 5'untanslated region (5'UTR) is different between them. This is significant because conserved regulatory RNA elements like upstream open reading frame (uORF) and putative internal ribosome entry site (IRES) were found in this region which have different combinations in each transcript variant and it may influence translational efficiency of them in normal or unusual environmental conditions like hypoxia. To indicate the transcription pattern of TSGA10 in breast cancer, expression of identified transcript variants was analyzed in 62 breast cancer samples. We found that TSGA10 tends to express variants with shorter 5'UTR and fewer uORF elements in breast cancer tissues. Our study demonstrates for the first time the expression of different TSGA10 transcript variants in testis and breast cancer tissues and provides a first clue to a role of TSGA10 5'UTR in regulation of translation in unusual environmental conditions like hypoxia.

Abstract 2125: Cis-mediated regulation of mRNA translation initiation of p53 family members

Abstract The p53 family of transcription factors comprises several proteins produced by three genes, TP63, TP63 and TP73, through alternative promoters and splicing, that exhibit critical functions for cell homeostasis and act as prominent tumor suppressors. The p53 pathway is often activated in stress conditions during which the global cap-dependent translation is inhibited. Although p53 protein is highly regulated at post-translational level, cells maintain continuous p53 protein synthesis also taking advantage of the presence of two Internal Ribosome Entry Sites (IRESs) elements residing within the p53 mRNA. The first IRES is located in the 5′UTR of the full-length isoform, the second is located into the protein-coding region and mediates the translation of a ΔN-p53 isoform. IRES elements are complex RNA structural elements in 5′UTRs that can mediate cap-independent initiation of translation under stress conditions. Even though an IRES activity has been already established for p53, no studies are available for p63 and p73 transcripts. This work is mainly focused on the regulation of translation initiation efficiency for the p53 family members p63 and p73. For this purpose, we have cloned the 5′UTR from human full-length p53 (as positive control), TA-p63, TA*-p63, TA-p73 and ΔN-p63 and ΔN-p73 transcript isoforms in a bicistronic reporter construct (named pRuF, where R stands for Renilla reniformis luciferase cDNA, u for 5′UTR tested and F for Firefly luciferase cDNA). The c-Myc and p16 or β-globin and β-actin 5′UTRs were included as positive or negative controls. We performed gene reporter assays in MCF7 and HCT116 p53 wild-type cancer cell lines and in their derivative clones with reduced (shp53) or null (p53-/-) p53 expression. It has been previously demonstrated that p53, through transcriptional repression of fibrillarin (FBL) is able to negatively impact on rRNA methylation pattern and IRES-dependent translation, hence p53 status can be an important factor to unmask cap-independent translation initiation. Results showed that TA*-p63, TA-p73 and ΔN-p73 can potentially contain an IRES-like sequence in their 5′UTR. Translation stimulation was increased in MCF7 cells with p53 was knocked-down. Treatment with the mTOR inhibitors Rapamycin and Torin1, that inhibit cap-dependent translation, led to an enhanced relative activity of the putative IRES contained in TA-p73. Interestingly, differences in responses of the various 5′UTRs between the two cell lines were observed. Based on these initial observations, the identification of different and tissue specific RNA binding proteins targeting the distinct p63 and p73 5′UTRs will be pursued with the aim to dissect a mechanism regulating mRNA translation initiation that could play an important role in development as well as in cancer progression. Note: This abstract was not presented at the meeting. Citation Format: Alessandra Bisio, Alberto Inga. Cis-mediated regulation of mRNA translation initiation of p53 family members. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2125. doi:10.1158/1538-7445.AM2015-2125

Mapping of internal ribosome entry sites (IRES).

The dicistronic luciferase reporter gene system is the most common method to isolate and characterize internal ribosome entry sites (IRES). It is based on the expression of a dicistronic RNA comprising two independent reporter genes in 3' and 5'cistrons, and the putative IRES inserted into intercistronic region. The most convenient aspect of using Renilla and firefly luciferase genes is that both gene products can be detected in a single assay using Dual-Luciferase(®) Reporter Assay System from Promega. The Renilla luciferase coding sequence is often inserted into the 5'cistron and serves as internal control. It is translated cap dependently, as it is close to the cap structure at the 5' end. The 3'cistron located far downstream to the cap structure can only be translated by a cap-independent mechanism when the intercistronic sequence is capable of ribosome binding and re-initiation of translation. Expression level of the 3'cistron is usually normalized to the expression of 5'cistron to estimate the relative IRES activity of intercistronic sequences.

Hyperoxia-induced alterations in the pulmonary proteome of juvenile rats

ABSTRACTHigh inspired concentrations of oxygen (hyperoxia) are often necessary to counteract tissue hypoxia during the treatment of ARDS. Reactive oxygen species generated by hyperoxic therapy may influence the expression of the pulmonary proteome and the application of discovery proteomics to the hyperoxic lung has the potential to divulge mechanisms regulating the expression of specific proteins integral to lung injury and repair. The present study examined the proteome derived from 30-day-old Sprague-Dawley rats exposed to room air (RA) and 95% O2 (Ox) for 24–72 hours using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with MALDI-ToF/ToF mass spectrometry. A total of 870 protein spots were visualized by 2D-DIGE across all gels. Mass spectral analysis identified 51 proteins representing 187 of the 214 significantly altered spots. Molecular and cellular function analysis grouped the identified proteins into free radical scavenging, cell death, cell-to-cell signaling, and cellular movement categories. The majority of the differences in the protein spots between RA and Ox occurred at 72 hours, with albumin, annexin A6 (AnxA6), and transferrin being increased, and mitochondrial Lon peptidase 1 being decreased by at least 20%. In Ox animals, AnxA6 protein expression increased three-fold without an increase in mRNA expression. Bioinformatic analysis of the AnxA6 transcript revealed the presence of a putative internal ribosome entry site within the 5′-untranslated region. These findings indicate that hyperoxia induces significant alterations in the pulmonary proteome which are temporally related. In addition, hyperoxia selectively enhances the expression of some proteins whose transcripts contain sequence motifs, which impart translational regulation.

Natural Occurrence and Characterization of Two Internal Ribosome Entry Site Elements in a Novel Virus, Canine Picodicistrovirus, in the Picornavirus-Like Superfamily

Dicistroviridae and Picornaviridae are two phylogenetically related families of positive-sense single-stranded RNA viruses in the picornavirus-like superfamily with similar gene contents but different genome organizations and hosts. In a surveillance study involving 1,472 samples from 368 dogs over a 22-month period, we identified a novel picornavirus-like virus from 47 fecal and urine samples by the use of reverse transcription-PCR (RT-PCR). Sequencing and phylogenetic analysis of three complete genomes revealed that, although it seemed that the virus was most closely related to other picornaviruses, P1, P2, and P3 of the virus possessed very low amino acid identities of <30% to those of all other known picornaviruses and that the amino acid identities between the 3D(pol) and 2C of the virus and the RNA-dependent RNA polymerases and helicases of all other picornaviruses were <35%. Distinct from other picornaviruses, the genomes of the virus contain two putative internal ribosome entry sites (IRESs) and two open reading frames, encoding two polyprotein precursors (844 and 1,406 amino acids), separated by an intergenic region (IGR) of 588 bases. A dual-luciferase activity assay using DNA and RNA transfection revealed that both IRESs were functional. Quantitative RT-PCR showed that numbers of viral RNAs ranged from 7.55 × 10(6) to 1.26 × 10(9) copies/ml of urine and 1.82 × 10(6) to 4.97 × 10(10) copies/ml of fecal sample. This is the first report of the natural occurrence of two functional IRESs in nondicistroviruses. Based on our results, we have proposed a novel species, canine picodicistrovirus (CPDV), to describe this novel member of the picornavirus-like superfamily, which could represent a novel family of viruses.

The duck hepatitis virus 5'-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences

Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.

Alternative Ways to Think about Cellular Internal Ribosome Entry

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of "cap-dependent" and "IRES-dependent" should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation.

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. We previously discovered an internal ribosome entry site (IRES) within the 5'-untranslated region of the human IGF1R mRNA, and identified and functionally characterized two individual RNA-binding proteins, HuR and hnRNP C, which bind the IGF1R 5'-UTR and differentially regulate IRES activity. Here we have developed and implemented a high-resolution northwestern profiling strategy to characterize, as a group, the full spectrum of sequence-specific RNA-binding proteins potentially regulating IGF1R translational efficiency through interaction with the 5'-untranslated sequence. The putative IGF1R IRES trans-activating factors (ITAFs) are a heterogeneous group of RNA-binding proteins including hnRNPs originating in the nucleus as well as factors tightly associated with ribosomes in the cytoplasm. The IGF1R ITAFs can be categorized into three distinct groups: (a) high molecular weight external ITAFs, which likely modulate the overall conformation of the 5'-untranslated region of the IGF1R mRNA and thereby the accessibility of the core functional IRES; (b) low molecular weight external ITAFs, which may function as general chaperones to unwind the RNA, and (c) internal ITAFs which may directly facilitate or inhibit the fundamental process of ribosome recruitment to the IRES. We observe dramatic changes in the northwestern profile of non-malignant breast cells downregulating IGF1R expression in association with acinar differentiation in 3-D culture. Most importantly, we are able to assess the RNA-binding activities of these putative translation-regulatory proteins in primary human breast surgical specimens, and begin to discern positive correlations between individual ITAFs and the malignant phenotype. Together with our previous findings, these new data provide further evidence that pathological dysregulation of IGF1R translational control may contribute to development and progression of human breast cancer, and breast metastasis in particular.

The XIAP IRES activates 3′ cistron expression by inducing production of monocistronic mRNA in the βgal/CAT bicistronic reporter system

X-chromosome linked inhibitor of apoptosis (XIAP) mRNA has been proposed to bear a stress-activated internal ribosome entry site (IRES) that stimulates translation under conditions that inhibit cap-dependent initiation. However, several reports have indicated that the strong activity of the XIAP IRES in certain bicistronic reporter assay systems stems from production of unintended monocistronic transcripts through splicing or cryptic promoter activity. Here we extend these findings by providing evidence that the XIAP IRES similarly provokes the production of monocistronic mRNA encompassing the 3' cistron in the betagal/CAT bicistronic reporter plasmid that was originally used to identify and characterize this putative IRES, through cryptic promoter activity.

Alternative Translation of OCT4 by an Internal Ribosome Entry Site and its Novel Function in Stress Response

OCT4 is a pivotal transcription factor in maintaining the pluripotency and self-renewal capacities of embryonic stem (ES) cells. Human OCT4 can generate two isoforms by alternative splicing, termed OCT4A and OCT4B. OCT4A confers the stemness properties of ES cells, whereas the function of OCT4B is unknown. We present here the diverse protein products and a novel function of OCT4 gene. A single OCT4B mRNA can encode three isoforms by alternative translation initiation at AUG and CUG start codons, respectively. A putative internal ribosome entry site (IRES) has been identified in OCT4B mRNA accounting for the translation mechanism. The OCT4B-190 is upregulated under stress conditions and it may protect cell against apoptosis under stress. This work evokes the significance to distinguish the biological function of the protein products of OCT4. The OCT4 gene, by the regulation of alternative splicing and alternative translation initiation, may carry out more crucial roles in many biological events.

Hypoxia-mediated Selective mRNA Translation by an Internal Ribosome Entry Site-independent Mechanism

Although it is advantageous for hypoxic cells to inhibit protein synthesis and conserve energy, it is also important to translate mRNAs critical for adaptive responses to hypoxic stress. Because internal ribosome entry sites (IRES) have been postulated to mediate this preferential synthesis, we analyzed the 5 '-untranslated regions from a panel of stress-regulated mRNAs for m(7)GTP cap-independent translation and identified putative IRES elements in encephalomyocarditis virus, vascular endothelial growth factor, hypoxia-inducible factors (HIFs) 1alpha and 2alpha, glucose transporter-like protein 1, p57(Kip2), La, BiP, and triose phosphate isomerase transcripts. However, when capped and polyadenylated dicistronic RNAs were synthesized in vitro and transfected into cells, cellular IRES-mediated translation accounted for less than 1% that of the level of cap-dependent translation. Moreover, hypoxic stress failed to activate cap-independent synthesis, indicating that it is unlikely that this is the primary mechanism for the maintenance of the translation of these mRNAs under low O(2). Furthermore, although HIF-1alpha is frequently cited as an example of an mRNA that is preferentially translated, we demonstrate that under different levels and durations of hypoxic stress, changes in newly synthesized HIF-1alpha and beta-actin protein levels mirror alterations in corresponding mRNA abundance. In addition, our data suggest that cyclin-dependent kinase inhibitor p57(Kip2) and vascular endothelial growth factor mRNAs are selectively translated by an IRES-independent mechanism under hypoxic stress.

Splicing mediates the activity of four putative cellular internal ribosome entry sites

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.

IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

BackgroundChanges of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.ResultsConnexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus in vivo and in vitro.ConclusionOur results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.