LucR, the first cistron, provides the level of cap-dependent translation. IRESes constitute powerful tools as they allow the expression of several genes from the same transcription unit. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 −/− osteosarcoma and neuroblastoma cell lines. nd, not detected. Given the involvement of FGF-2 in cancer and injury repair, such data suggest a pivotal role of the IRES in the subtle regulation of FGF-2 expression in vivo. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. In contrast, as observed from transfection experiments, FGF-2 IRES functions in most tissue culture cell types ex vivo. FGF-2 IRES was weakly expressed in most organs, except the testis (up to 40.7) and the brain, where it presented exceptionally high activity (up to 454). 1998; Zhou et al. The study of cells is called cell biology, cellular biology, or cytology.. This IRES is constitutively active in transformed cells and could account for the deregulated expression of CUG1, -2, and -3–initiated FGF-2 isoforms (HMW FGF-2; Galy et al. The effect of several ribosome-inactivating proteins (saporin 6, dianthin 32, pokeweed antiviral protein from seeds, gelonin, bryodin-R, and momordin) on primary cultures of human trophoblasts and human embryonal fibroblasts and on choriocarcinoma (JAR and BeWo) and ovarian carcinoma (TG) cell lines was studied. We hypothesize from these results that retroviral IRESes and cellular IRESes, which both belong to capped and polyadenylated transcripts, would present stringent tissue specificity, whereas the activity spectrum of picornavirus IRESes, which in most cases belong to uncapped mRNA, would be broader. Interestingly, FGF-2 proteins were observed in tissues such as the brain where the FGF-2 mRNA could not be detected, even by RT-PCR (Coffin et al. Concurrent host-parasite ribosome profiling. 1998). FGF-2 and EMCV IRESes activities in transgenic mice embryos. FGF-5 RNA synthesized in vitro can be translated in rabbit reticulocyte lysates to yield a 29,500-Da protein, which is consistent with the molecular weight predicted from the coding sequence. Interestingly, IRES elements are found in the mRNAs of several survival factors, oncogenes and proteins … We show in this study that EMCV IRES functions in a ubiquitous manner not only in the embryos, but also in most organs of adult transgenic mice. Virology. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. IRES-dependent regulation of FGF-2 mRNA translation in pathophysiological conditions in the mouse. COS-7 (accession number for ATCC, CRL 1654) is a monkey kidney cell transformed by SV-40 large T antigen. NIH This site needs JavaScript to work properly. Autophagosomes engulf and degrade cellular components in lysosomes. The two luciferase genes, Renilla luciferase (LucR) and firefly luciferase (LucF), are controlled by the cytomegalovirus promoter (CMV) and separated by either a hairpin or the encephalomyocarditis virus (EMCV) IRES, for pCRHL and pCREL, respectively (see Fig. The PCR was performed with the primer couples, 5′-GATTACCAGGGATTTCAGTCG-3′ and 5′-CTGAAGGGACTGTAAAAACAGC-3′, 5′-CCACATATTGAGCCAGTAGC-3′ and 5′-CCATGATAATGTTGGACGAC-3′, to amplify the LucF or LucR DNA sequences, respectively. The researchers also found that ribosomes derived from yeast and human fibroblast cells also have the same initializing activity. Furthermore, the IRES of an avian retrovirus shows considerable activity in multipotent neural cells and in their glial and neuronal progeny (Derrington et al. It regulates gap junction communications in astroglial cells (Reuss et al. The positive control (C+) was performed with an in vitro–transcribed bicistronic RFL mRNA. It is synthesized as a 155 amino acid polypeptide, whose mature form is a non-glycosylated 17-18 kDa protein.Fibroblast growth factor protein was first purified in 1975, but soon afterwards others using different conditions isolated acidic FGF, … in an amplification of ribosome occupancy at the start codon (Supplementary Fig. 4-fold FC is used as a cutoff, and proteins above this cutoff are marked. A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework for animal tissues, and plays a critical role in wound healing. Using a recently reported method to isolate lysosomes, Wyant et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. With respect to the mean value of the FANC pathway– proficient cells (12%; red dotted line in Fig. IRESes, which allow cap-independent translation initiation, have been discovered in several cellular capped mRNAs during the last few years. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. These observations may be related to another study performed in transgenic flies indicating that the IRES contained in the 5′ untranslated regions of antennapedia and ultrabithorax exhibited a high degree of developmental regulation and were not functionally equivalent (Ye et al. New pathologic mechanisms in nucleotide repeat expansion disorders. 2020 Jun 23;11(6):686. doi: 10.3390/genes11060686. As shown in Fig. These data also show that the human FGF-2 IRES activity is more tissue-specific than species-specific. This suggested that the tissue origin of the cell line might be one parameter conditioning FGF-2 IRES activity. IRES activities are represented by histograms and were measured by calculating the LucF/LucR ratio (Table). 2, the FGF-2 IRES showed a large spectrum of activity since it was active in all the different mammalian cell types tested. 2019 Oct 29;9(11):665. doi: 10.3390/biom9110665. LucF, the second cistron, is expressed in proportion to the IRES activity. Clipboard, Search History, and several other advanced features are temporarily unavailable. Having more ribosomes helps growing cells cope with the demand for making new protein, and ribosomes themselves can account for up to 40% of the total cel-lular mass. Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes. 1995), suggesting conservation of the regulatory mechanisms between species. In contrast, EMCV IRES activity was high in all the tissues of the two REL transgenic lines embryos tested, except in the liver of E16 embryos (Fig. Indeed, we find high IRES activity in the brain and weaker activity in the testes, ovaries and lung, whereas all the other tested organs show no significant IRES activity, even those in which the CMV promoter is active. Jurkat (accession number for ATCC, TIB-152) is a human acute T cell leukemia. In those transgenic animals, the pattern of expression of the different human isoforms was highly tissue-specific and corresponded to that of the endogenous FGF-2 isoforms. Several cis-acting elements located within the FGF-2 mRNA leader sequence are involved in the control of FGF-2 translation (Prats et al. 1997). In summary, these data indicate that the FGF-2 IRES is subjected to stringent regulation in the adult, and that its activity is strongly upregulated in the brain. Recently, we discovered that ribosome is involved in the regulation of cellular transdifferentiation of human dermal fibroblasts (HDFs). The bicistronic vector strategy, which was previously used to demonstrate the existence of the two vascular endothelial growth factor IRESes (Huez et al. Neurobiol Dis. 1998). They contain the CMV promoter controlling the expression of a bicistronic LucR-LucF mRNA. Our strategy was to express a bicistronic mRNA encoding two different luciferase reporter genes, the second being under the control of the human FGF-2 IRES. 3, right). ABAE is an adult bovine aortic endothelial primary cell (Couderc et al. 1998). SK-Hep-1 (accession number for ATCC, HTB 52) is a human liver adenocarcinoma of endothelial origin; SK-N-AS (accession number for ECACC, 94092302) and SK-N-BE (accession number for EACC, 95011815) are human neuroblastoma. Biochem Soc Trans. and differentiation. 3. The results are shown for each of the two independent transgenic lines expressing one or other bicistronic construct pCRFL (FGF-2) or pCREL (EMCV), as means ± SEM of experiments that were performed 3–10 times. This allows for an mRNA-resolved transcriptome-wide view of scanning ribosomes in human cells. The use of such an assay, based on dual highly sensitive reporters, allows rapid and concomitant quantitation of reporter activity. As indicated in Fig. USA.gov. Indeed, it is not only active in human cultured cells, but also in hamster, monkey, and mouse cells or tissues, suggesting that specific cellular trans-acting factors are able to recognize FGF-2 IRES independently of their origin. 2003 Oct 10;278(41):39330-6. doi: 10.1074/jbc.M305580200. 293 (accession number for ATCC, CRL-1573) is a human kidney epithelial cell, transformed with adenovirus 5. This experiment enabled us to check the LucF/LucR ratios at the RNA level in the different organs. The supernatant was centrifuged for 15 min at 13,000 rpm at 4°C, and the last supernatant was used for luminescence dosage (30 μl). L. Créancier was financed first by the European Commission BIOTECH program, and then by Retina France. However, the bacterium-intrinsic cellular- transdifferentiation factor remained unknown. Ribosome Incorporation Into Somatic Cells Promotes Lineage Transdifferentiation Towards Multipotency - PubMed Recently, we reported that bacterial incorporation induces cellular transdifferentiation of human fibroblasts. The in vivo specificity of this activity contrasted strikingly with the relatively ubiquitous expression of viral EMCV IRES. These data show that FGF-2 IRES is active in the early embryo, but is strongly regulated in a tissue-specific manner. 13 Acute reductions of OGFOD1 in mammalian cells alter translational fidelity and increase the formation of mRNA-containing stress granules. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Initiation of translation of human rhinovirus RNA: mapping the internal ribosome entry site. In vitro in-corporation of ribosomes into HDFs arrests cell proliferation and induces the formation of cell clusters, that differentiate into three germ layer derived cells upon IRES activity was determined from the ratio of second cistron to first cistron expression. The lines expressing pCREL (right) are RELB (gray boxes) and RELA (black boxes). 48 h after transfection, cell lysates were prepared for luminescence activity as previously described (Huez et al. Cells were harvested 48 h after transfection, and the luciferase activities present in cell extracts were measured. Growth Factors. 1995). This is in agreement with a recent report that demonstrates a quasi-ubiquitous activity of the IRES of another picornavirus, Theiler's murine encephalomyelitis virus, in young and adult transgenic mice (Shaw-Jackson and Michiels 1999). The results are shown for each of the two independent transgenic lines, expressing one or other bicistronic construct pCRFL (FGF-2) or pCREL (EMCV), as means ± SEM of experiments that were performed 3–10 times. It is expected to reflect the activity of CMV sequences in the tissues or cells and, thus, to be proportional to the amount of bicistronic mRNA. Finally, the stringent tissue specificity of a given cellular IRES might be very useful as a biotechnological tool to drive specific gene expression in a particular tissue. 1999). 1997; Meisinger and Grothe 1997). Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. FGF-2 is also implicated in wound-healing processes (Bikfalvi et al. [Article in Russian] Zharskaia OO(1), Barsukova AS, Medzhidova AA, Fedorova NE, Kushch AA, Zatsepina OV. 1992. Total RNA was extracted from RFL12 or RELA transgenic mice brain (B), tongue (To), or testis (Te), and the level of LucF and LucR cistrons was analyzed by RT-PCR (as described in Materials and Methods). Vagner S(1), Gensac MC, Maret A, Bayard F, Amalric F, Prats H, Prats AC. This work was supported by grants from the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, the Conseil Régional Midi-Pyrénées, the European Commission BIOTECH program (contrat 94199-181), and Rhone-Poulenc-Rorer (now Aventis). As shown in Fig. 1992 Jun; 188 (2):685–696. 1997). 80S human ribosome structure with the positions of uS3 (green), uS20 (orange), uL16 (dark blue), mRNA (red), E-site tRNA (dark gray), and EEF2 (black) are indicated. 1990;44(1-4):278-91. doi: 10.1159/000468765. 1995). 1998). FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system. The tissue specificity that we observed is strongly reminiscent of the proposed role of FGF-2 in embryogenesis and the adult CNS (Eckenstein 1994; Slack and Isaacs 1994; Houle and Ye 1999). 12:4807], Differential expression of FGF-2 isoforms in the rat adrenal medulla during postnatal development in vivo, Structure and expression of a novel human FGF, FGF-19, expressed in the fetal brain, Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2, Cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor, High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons, Selective expression of high molecular weight basic fibroblast growth factor confers a unique phenotype to NIH 3T3 cells, Gap junction uncoupling of neonatal rat astroglial cultures does not affect their expression of fibroblast growth factor 2, Absence of internal ribosome entry site-mediated tissue specificity in the translation of a bicistronic transgene, The role of fibroblast growth factors in early Xenopus development, Expression of human FGF-2 is post-transcriptionally controlled by a unique destabilization element present in the mRNA 3′ untranslated region between alternative polyadenylation sites, Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes, Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells, Ultrabithorax and antennapedia 5′ untranslated regions promote developmentally regulated internal translation initiation, Fibroblast growth factor 2 control of vascular tone, This site uses cookies. , Pineda B, Rodrigues P, Tormo E, Terol MJ, P.! 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