IRES Region Research Articles

LncRNA CTBP1-DT-encoded microprotein DDUP sustains DNA damage response signalling to trigger dual DNA repair mechanisms.

Sustaining DNA damage response (DDR) signalling via retention of DDR factors at damaged sites is important for transmitting damage-sensing and repair signals. Herein, we found that DNA damage provoked the association of ribosomes with IRES region in lncRNA CTBP1-DT, which overcame the negative effect of upstream open reading frames (uORFs), and elicited the novel microprotein DNA damage-upregulated protein (DDUP) translation via a cap-independent translation mechanism. Activated ATR kinase-mediated phosphorylation of DDUP induced a drastic ‘dense-to-loose’ conformational change, which sustained the RAD18/RAD51C and RAD18/PCNA complex at damaged sites and initiated RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair mechanisms. Importantly, treatment with ATR inhibitor abolished the effect of DDUP on chromatin retention of RAD51C and PCNA, thereby leading to hypersensitivity of cancer cells to DNA-damaging chemotherapeutics. Taken together, our results uncover a plausible mechanism underlying the DDR sustaining and might represent an attractive therapeutic strategy in improvement of DNA damage-based anticancer therapies.

Simulated vector transmission differentially influences dynamics of two viral variants of deformed wing virus in honey bees (Apis mellifera).

Understanding how vectors alter the interactions between viruses and their hosts is a fundamental question in virology and disease ecology. In honey bees, transmission of deformed wing virus (DWV) by parasitic Varroa mites has been associated with elevated disease and host mortality, and Varroa transmission has been hypothesized to lead to increased viral titres or select for more virulent variants. Here, we mimicked Varroa transmission by serially passaging a mixed population of two DWV variants, A and B, by injection through in vitro reared honey bee pupae and tracking these viral populations through five passages. The DWV-A and DWV-B variant proportions shifted dynamically through passaging, with DWV-B outcompeting DWV-A after one passage, but levels of both variants becoming equivalent by Passage 5. Sequencing analysis revealed a dominant, recombinant DWV-B strain (DWV-A derived 5′ IRES region with the rest of the genome DWV-B), with low nucleotide diversity that decreased through passaging. DWV-A populations had higher nucleotide diversity compared to DWV-B, but this also decreased through passaging. Selection signatures were found across functional regions of the DWV-A and DWV-B genomes, including amino acid mutations in the putative capsid protein region. Simulated vector transmission differentially impacted two closely related viral variants which could influence viral interactions with the host, demonstrating surprising plasticity in vector-host-viral dynamics.

PTBP3 contributes to colorectal cancer growth and metastasis via translational activation of HIF-1\u03b1

BackgroundColorectal cancer (CRC) remains the fourth most common cause of cancer-related mortality worldwide. We aimed to identify key molecules and signalling pathways mediating CRC growth and metastasis. Polypyrimidine tract-binding protein 3 (PTBP3) is a member of PTB family. A prooncogenic role for PTBP3 has also been discovered in several kinds of tumors. However, the expression and biological functions of the PTBP3 are still unknown in CRC.MethodsWe analysed the expression levels of PTBP3 using tissue microarray containing 568 CRC tissues and corresponding non-tumor adjacent tissues. The correlations between the PTBP3 expression level and clinicopathological features were evaluated using the chi-square test. The functional characterization for the role and molecular mechanism of PTBP3 in CRC was investigated through a series of in vitro and in vivo experiments.ResultsWe showed that PTBP3 expression was increased in human CRC, and high PTBP3 expression was correlated with poor five-year overall survival and disease-free survival. Moreover, PTBP3 promoted tumor cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo. PTBP3 enhanced HIF-1α protein expression by directly binding to the 5′UTR HIF-1α mRNA and activated translation of HIF-1α. Furthermore, HIF-1α was responsible for PTBP3-induced cell migration and invasion.ConclusionsPTBP3 appears to be a novel oncogene of CRC through binding to the IRES region of HIF-1α mRNA, which regulates HIF-1α translation. PTBP3 can serve as a promising predictive biomarker for recurrence and prognosis in patients with CRC.

Beyond sites 1 and 2, miR-122 target sites in the HCV genome have negligible contributions to HCV RNA accumulation in cell culture.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to two adjacent sites (S1 and S2) located at the 5' end of the viral RNA genome. This interaction promotes HCV RNA accumulation by stabilising the viral RNA and resulting in alteration of the secondary structure of the viral genome. In addition to S1 and S2, the HCV genome contains several other putative miR-122 binding sites, one in the IRES region, three in the NS5B coding region, and one in the 3' UTR. We investigated and compared the relative contributions of the S1, S2, IRES, NS5B (NS5B.1, 2 and 3) and 3' UTR sites on protein expression, viral RNA accumulation, and infectious particle production by mutational analysis and supplementation with compensatory mutant miR-122 molecules. We found that mutations predicted to alter miR-122 binding at the IRES and NS5B.2 sites lead to reductions in HCV core protein expression and viral RNA accumulation; with a concomitant decrease in viral particle production for the NS5B.2 mutant. However, supplementation of miR-122 molecules with compensatory mutations did not rescue these site mutants to wild-type levels, suggesting that mutation of these sequences likely disrupts an additional interaction important to the HCV life cycle, beyond direct interactions with miR-122. Thus, S1 and S2 play a predominant role in viral RNA accumulation, while miR-122 interactions with the IRES, NS5B and 3' UTR regions have negligible contributions to viral protein expression, viral RNA accumulation, and infectious particle production.

Unexplained isolated hyperferritinemia without iron overload.

Although hyperferritinemia may be reflective of elevated total body iron stores, there are conditions in which ferritin levels are disproportionately elevated relative to iron status. Autosomal dominant forms of hyperferritinemia due to mutations in the L-ferritin IRE or in A helix of L-ferritin gene have been described, however cases of isolated hyperferritinemia still remain unsolved. We describe 12 Italian subjects with unexplained isolated hyperferritinemia (UIH). Four probands have affected siblings, but no affected parents or offspring. Sequencing analyses did not identify casual mutations in ferritin gene or IRE regions. These patients had normal levels of intracellular ferritin protein and mRNA in peripheral blood cells excluding pathological ferritin production at transcriptional and post-transcriptional level. In contrast with individuals with benign hyperferritinemia caused by mutations affecting the ferritin A helix, low rather than high glycosylation of serum ferritin was observed in our UIH subjects compared with controls. These findings suggest that subjects with UIH have a previously undescribed form of hyperferritinemia possibly attributable to increased cellular ferritin secretion and/or decreased serum ferritin clearance. The cause remains to be defined and we can only speculate the existence of mutations in gene/s not directly implicated in iron metabolism that could affect ferritin turnover including ferritin secretion.

Discovery of Dual Inhibitors of MDM2 and XIAP for Cancer Treatment

MDM2 and XIAP are mutually regulated. Binding of MDM2 RING protein to the IRES region on XIAP mRNA results in MDM2 protein stabilization and enhanced XIAP translation. In this study, we developed a protein-RNA fluorescence polarization (FP) assay for high-throughput screening (HTS) of chemical libraries. Our FP-HTS identified eight inhibitors that blocked the MDM2 protein-XIAP RNA interaction, leading to MDM2 degradation. The compound-induced MDM2 downregulation resulted not only in inhibition of XIAP expression, but also in activation of p53, which contributed to cancer cell apoptosis invitro and inhibition of cancer cell proliferation invivo. Importantly, one of the MDM2/XIAP inhibitors, MX69, showed minimal inhibitory effect on normal human hematopoiesis invitro and was very well tolerated in animal models.

Busca por Hepacivirus semelhante ao vírus da hepatite C, em primatas não humanos

Hepacivirus genus comprises hepatitis C virus (HCV), but a proposal to include other viruses, originated from dogs, horses, rodents, and GB virus B (GBV-B), is being made. HCV, or any other virus phylogenetically related to it, has not been identified in wild nonhuman primates, differently from HBV-like viruses. HCV infections in humans are severe, therefore a search for Hepacivirus in other animals is needed for they can cross the evolutionary barrier causing infections in humans. This study tried to identify a nonhuman primate origin for Hepacivirus. Primers have been designed, which bind on HCV IRES region, resulting a final product of 188 bp. A total of 1,054 samples collected from 36 nonhuman primate species were analyzed, of which just 12 presented defined bands and were negative for Hepacivirus after sequencing. In evolutionary analysis, GB virus C in a phylogenetic position is the most distant from HCV, while GBV-B is the nearest. Although HCV-related Hepacivirus is not found in nonhuman primates, its existence cannot be denied. Studies involving other species, such as GB viruses and possible new Hepacivirus, can solve the evolutionary process that has allowed HCV infect humans, since they seem to be the link between HCV and viral species found in wild animals.

Genomic comparison of foot-and-mouth disease virus R strain and its chick-passaged attenuated strain

The present study examined the genomic differences between foot-and-mouth disease virus (FMDV) R strain and its attenuated, chick-passaged (R 304) strain. Eleven pairs of primers were used to amplify the complete genome of FMDV R and R 304 by RT-PCR. Each fragment was cloned into pMD18-T vector and sequenced. Nucleotide analyses showed that the genome encoding regions of R and R 304 strains open reading frame (ORF) were both 6966 nucleotides (nt) in length, encoding 2322 amino acids. One hundred and ten nucleotides or 32 amino acids were found to be mutated most frequently were in the 3A gene. The next highest rates of mutation were observed in the LP and 1D genes. No mutations were found in either the 2A or 2C genes. The length of 5′IRES region and 3′UTR were 450 nt and 94 nt, respectively. The 5′IRES region and 3′UTR had only 4 nt and 3 nt mutation, respectively after attenuation. The R 304 poly(A) tail length of 18 nt, while that of the R strain was 30 nt. This result demonstrated the primary genomic changes of a FMDV and its attenuated strain, which has important implications in understanding the molecular epidemiology and functional genomics of FMDV.

Structural Domains of the 3′-Terminal Sequence of the Hepatitis C Virus Replicative Strand

Here we present the results of a structural analysis of the 3'-terminal region of the replicative strand of hepatitis C virus (HCV), IRES(-), by the Pb (2+)-induced cleavage approach and partial digestion with T1 ribonuclease. Oligoribonucleotides that represent selected domains of the earlier proposed in the literature secondary structure models of this region were also synthesized, their structures were analyzed in solution, and the results were compared to those obtained with the full-length molecule. Such "structural fingerprinting" gave better insight into the structure of the IRES(-) region. We showed that in the case of the IRES(-) fragment, which consists of 374 nucleotides, its three domains, D3 (nucleotides 1-104), DM (nucleotides 105-222), and D5 (nucleotides 223-374), independently fold on one another. However, when the IRES(-) molecule is extended by 25 nucleotides of the upstream viral sequence, domains D3 and DM fold autonomously, but a part of domain D5 interacts with that additional RNA stretch. Analysis in silico suggests that similar interactions involving the IRES(-) region and upstream sequences are also possible in other fragments of viral RNA, several hundreds of nucleotides in length. The results of experimental probing are supported by secondary structure predictions in silico and phylogenetic analysis.

Development and comparison of genome detection assays for the diagnosis of foot-and-mouth disease suspected clinical samples

Detection of foot-and-mouth disease virus (FMDV) from clinical specimens by conventional sandwich enzyme-linked immunosorbent assay (ELISA) and virus isolation in cell culture is often compromised owing to limited sensitivity and inactivation during transit, respectively. A RT-PCR (oligoprobing) ELISA in both solid and aqueous phase hybridization formats targeting an across serotype conserved site at 3C–3D region was developed and its effectiveness was compared with that of the known targets at the IRES region. A non-isotopic RNA dot hybridization assay with colorimetric detection targeting both the IRES and the 3D region were also validated, which is capable of handling high throughput samples with ease. RT-PCR (oligoprobing) ELISA and dot hybridization assay showed 1000- and 10-fold greater sensitivity than the sandwich ELISA, respectively. Robustness of these diagnostic methods was explored by examining on sandwich ELISA-negative clinical samples. Both the assays developed in the present study were able to detect viral genomes in samples undetectable by conventional ELISA, thereby demonstrating ‘proof of sensitivity’. Although the potential of these assays for providing definitive diagnosis in carrier hosts and in species where clinical disease is inapparent remains to be examined, nevertheless these assays can be adapted for comprehensive surveillance of foot-and-mouth disease in India.

RNAi expression mediated inhibition of HCV replication

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Oxidative Stress Induces Activation of a Cytosolic Protein Responsible for Control of Iron Uptake

A cytosolic protein, named iron-responsive element-binding protein (IRE-BP), is sensitive to cellular iron concentration. At low cytosolic iron level, IRE-BP is activated and binds to stem-loop untranslated regions (IRE regions) of transferrin and ferritin mRNAs, activating and inhibiting their translations, respectively. This concerted mechanism permits a fine control of iron homeostasis in the cell. The activity of IRE-BP can be measured by its binding to IRE regions, using a protein band-shift electrophoretic assay. Damage to cells by oxidative stress is known to be mediated by iron. We observed that IRE-BP is rapidly activated by exposure of V79 Chinese hamster ovary cells to H2O2. However, if cell extracts are exposed to H2O2 IRE-BP activation is not observed, Therefore, the activation is not a direct consequence of the H2O2 attack to IRE-BP. The in vivo IRE-BP-activation by H2O2 is not prevented by hydroxyl radical scavengers or by the iron chelator 1,10-phenanthroline, indicating that Fenton reaction is not involved in the process, In fact, simultaneous exposure of cells to H2O2 and 1,10-phenanthroline produces an even stronger activation than exposure to H2O2 alone. The interpretation of the mechanism of IRE-BP activation by oxidative stress is hampered by the fact that the mechanism of IRE-BP modulation by cytosolic iron has not been established. It has been recently shown that the iron-sulfur cluster in IRE-BP must be completely disassembled in order for activation to occur and that this is triggered by low iron in the cell. It is likely that IRE-BP senses Fe(II) and that its oxidation to Fe(III) by H2O2 or chelation by 1,10-phenanthroline set up a program for increasing iron uptake. The physiological consequences of this activation still has to be assessed.

Structure and function of IREs, the noncoding mRNA sequences regulating synthesis of ferritin, transferrin receptor and (erythroid) 5-aminolevulinate synthase.

The synthesis of least three proteins involved in iron metabolism is coordinately regulated in animals through noncoding sequences in mRNA, the IREs; the transcription of the genes encoding the proteins are also regulated. Cellular iron is the best known effector of changes in regulation of mRNA with IREs. A hairpin loop is the secondary structure of IRES which conserve the hairpin loop sequence, CAGUGU/C. However, variable stem sequences, apparently related to mRNA-specific function, create a family of IRE regulatory sequences. At least three types of proteins recognize IRE regions: (1) Nucleases which degrade mRNAs with 3' noncoding IRES; the IRE/IRE-BP stabilizes mRNAs with 3' noncoding IRES (transferrin receptor mRNA). (2) Initiation factors/ribosomes; the IRE/IRE-BP blocks ribosome binding of mRNAs with 5' noncoding IREs (ferritin, eALAS mRNAs). (3) Initiation factors to enhance translation (ferritin mRNA) when the IRE-BP does not bind; the ferritin IRE is thus both a negative and positive control element depending on which type of protein is bound. The IRE in ferritin mRNA is the most studied IRE to date. Site-directed mutagenesis shows that sites throughout the IRE alter negative control and IRE-BP binding reflecting the fact that the footprint of the IRE-BP is over the entire IRE. Base paired flanking regions (FL) which are ferritin IRE specific, enhance the effects of IRE-BP binding on negative control. Positive control is altered by modifying the single sites in stem/internal loop but not in the hairpin loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Internal reflection spectroscopy applied to the analysis of adhesive tapes

Internal Reflection Spectroscopy, IRS, provides a powerful technique for the in situ investigation and characterization of the functional layers of an adhesive tape. An adhesive tape is a multicomponent system comprised of several functional layers, each layer being a single polymer or a mixture of polymers, sometimes blended with mineral components. The internal reflection techniques applied to tapes may also be extended to the analysis of similar types of polymers and polymer blends. To optimize the spectral information obtained using multiple internal reflection techniques, careful consideration must be given to the internal reflection equipment, particularly the internal reflection element, IRE, or sampling plate. Guidelines for sampling and finding effective surface areas as well as most sensitive IRE regions are presented for some commercially available instrumentation. Some applications of IRS to adhesive tape analysis are presented, including qualitative identification of tape components using limited sample, fingerprinting of tape layers for storage and retrieval, reactivity studies of curing tape layers, migration studies, and identification of surface contaminants which alter the intended function of tape layers.

Roster of PGMTT Members Listed by IRE Regions and Sections, as of March 1, 1960

Roster of PGMTT Members Listed by IRE Regions and Sections, as of March 1, 1960

Roster of PGIE Members Listed by IRE Regions and Sections, As of June 19, 1959

Roster of PGIE Members Listed by IRE Regions and Sections, As of June 19, 1959

Roster of PGB Members Listed by IRE Regions and Sections as of January 30, 1959

Roster of PGB Members Listed by IRE Regions and Sections as of January 30, 1959