Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The human insulin receptor mRNA contains a functional internal ribosome entry segment.

ABSTRACT: Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.

SUBMITTER: Spriggs KA

PROVIDER: S-EPMC2761284 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site.

Project description:We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.

| S-EPMC114096 | biostudies-literature

A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site.

Project description:As the hormone gastrin promotes gastrointestinal (GI) cancer progression by triggering survival pathways, regulation of gastrin expression at the translational level was explored. Sequence within the 5' untranslated region of a gastrin transcript expressed in GI cancer cells was investigated, then cloned into a bicistronic vector upstream of firefly luciferase and transfected into a series of GI cancer cell lines. Firefly luciferase activity was measured relative to that of a cap-dependent Renilla luciferase. A gastrin transcript that was different from that described in Ensembl was expressed in GI cancer cells. Its transcription appears to be initiated within the region designated as the gene's first intron. In GI cancer cells transfected with the bicistronic construct, firefly luciferase activity increased 8-15-fold compared with the control vector, and there was a further induction of the signal (up to 25-fold) following exposure of the cells to genotoxic stress or hypoxia, suggesting that the sequence acts as an internal ribosome entry site. These data suggest that the gastrin transcript within GI cancer cells contains an internal ribosome entry site that may allow continued expression of gastrin peptides when normal translational mechanisms are inactive, such as in hypoxia, thereby promoting cancer cell survival.

| S-EPMC2391123 | biostudies-literature

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation.

Project description:Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.

| S-EPMC3783170 | biostudies-literature

Hypoxia-mediated selective mRNA translation by an internal ribosome entry site-independent mechanism.

Project description: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.

| S-EPMC2423241 | biostudies-literature

Identification of internal ribosome entry segment (IRES)-trans-acting factors for the Myc family of IRESs.

Project description:The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.

| S-EPMC2223313 | biostudies-literature

The 5' leader of the mRNA encoding the marek's disease virus serotype 1 pp14 protein contains an intronic internal ribosome entry site with allosteric properties.

Project description:We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.

| S-EPMC2786844 | biostudies-literature

Internal ribosome entry segment-mediated initiation of c-Myc protein synthesis following genotoxic stress.

Project description:Initiation of translation of the proto-oncogene c-myc can occur by either the cap-dependent scanning mechanism or by internal ribosome entry. The latter mechanism requires a complex RNA structural element that is located in the 5' untranslated region of c-myc, termed an internal ribosome entry segment (IRES). Recent work has shown that IRESs are used to maintain protein expression under conditions when cap-dependent translation initiation is compromised; for example, during mitosis, apoptosis and under conditions of cell stress, such as hypoxia or heat shock. Induction of genotoxic stress also results in a large reduction in global protein synthesis rates and therefore we investigated whether the c-myc IRES was active following DNA damage. As expected, in cells treated with either ethylmethane sulphonate or mitomycin C there was a large reduction in protein synthesis, although this was brought about by two different mechanisms. However, in each case the c-myc IRES was active and c-Myc protein expression was maintained. Finally we showed that the proteins required for this process are downstream of the p38 mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase (ERK)/MEK(MAPK/ERK kinase) signalling pathways, since pre-treatment of cells with inhibitors of these pathways before DNA damage is initiated inhibits both c-myc IRES activity and expression of c-Myc protein.

| S-EPMC1222134 | biostudies-other

In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites.

Project description:Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs.

| S-EPMC3167618 | biostudies-literature

Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1.

Project description:The 5'untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.

| S-EPMC3319624 | biostudies-literature

Deconstructing internal ribosome entry site elements: an update of structural motifs and functional divergences.

Project description:Beyond the general cap-dependent translation initiation, eukaryotic organisms use alternative mechanisms to initiate protein synthesis. Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of translation using a cap-independent mechanism. However, their lack of primary sequence and secondary RNA structure conservation, as well as the diversity of host factor requirement to recruit the ribosomal subunits, suggest distinct types of IRES elements. In spite of this heterogeneity, conserved motifs preserve sequences impacting on RNA structure and RNA-protein interactions important for IRES-driven translation. This conservation brings the question of whether IRES elements could consist of basic building blocks, which upon evolutionary selection result in functional elements with different properties. Although RNA-binding proteins (RBPs) perform a crucial role in the assembly of ribonucleoprotein complexes, the versatility and plasticity of RNA molecules, together with their high flexibility and dynamism, determines formation of macromolecular complexes in response to different signals. These properties rely on the presence of short RNA motifs, which operate as modular entities, and suggest that decomposition of IRES elements in short modules could help to understand the different mechanisms driven by these regulatory elements. Here we will review evidence suggesting that model IRES elements consist of the combination of short modules, providing sites of interaction for ribosome subunits, eIFs and RBPs, with implications for definition of criteria to identify novel IRES-like elements genome wide.

| S-EPMC6282068 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data