Effect of Low-Dose Persistent Organic Pollutants on Mitochondrial Function: Human and in Vitro Evidence.

BACKGROUND: Chronic exposure to low-dose persistent organic pollutants (POPs) can induce mitochondrial dysfunction. This study evaluated the association between serum POP concentrations and oxygen consumption rate (OCR) as a marker of mitochondrial function in humans and in vitro cells. METHODS: Serum concentrations of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were measured in 323 adults. The OCRs of platelets and peripheral blood mononuclear cells (PBMCs) were assessed in 20 mL of fresh blood using a Seahorse XF analyzer. Additionally, the in vitro effects of Arochlor-1254, ß-hexachlorocyclohexane, and p,p´-dichlorodiphenyltrichloroethane at concentrations of 0.1 pM to 100 nM were evaluated in human platelets, human PBMCs, and Jurkat T-cells. RESULTS: The association between serum POP concentrations and OCR differed depending on the cell type. As serum OCP concentrations increased, basal platelet OCR levels decreased significantly; according to the OCP quintiles of summary measure, they were 8.6, 9.6, 8.2, 8.0, and 7.1 pmol/min/µg (P trend=0.005). Notably, the basal PBMC OCR levels decreased remarkably as the serum PCB concentration increased. PBMC OCR levels were 46.5, 34.3, 29.1, 16.5, and 13.1 pmol/min/µg according to the PCB quintiles of summary measure (P trend <0.001), and this inverse association was consistently observed in all subgroups stratified by age, sex, obesity, type 2 diabetes mellitus, and hypertension, respectively. In vitro experimental studies have also demonstrated that chronic exposure to low-dose POPs could decrease OCR levels. CONCLUSION: The findings from human and in vitro studies suggest that chronic exposure to low-dose POPs can induce mitochondrial dysfunction by impairing oxidative phosphorylation.

Lifestyle of Patients with Alcoholic Liver Disease and Factors Leading to Hospital Readmission: A Prospective Observational Study.

The objective of this study was to clarify the lifestyle characteristics of patients with alcoholic liver disease (ALD) who were readmitted to the hospital, and to identify the background factors associated with these characteristics. This was a prospective observational study. Over a period of 3 months following hospital discharge, we conducted structured interviews to investigate the following five lifestyle characteristics based on our previous research: dietary intake, alcohol consumption or abstinence, psycho-emotional status, regularity of life habits, adherence to treatment. We also collected data on background factors from medical records and questionnaires. The analysis was performed using conceptual cluster matrices, with participants divided into two groups (at-home recovery and readmission). Lifestyle, health status, and background factors were compared between the two groups. Of the 34 patients with ALD recruited, 21 completed the one-month follow-up and were included in the analysis-14 patients were in the at-home recovery group and 7 in the readmission group. The at-home group's lifestyle was characterized by controlled alcohol consumption, but with maintenance of regular life and eating habits and adherence to treatment. In contrast, irregular eating habits (p=0.006) and the development of irregular life habits or the discontinuation of treatment very quickly after hospital discharge characterized the readmission group's lifestyle. Experiences of loss were a lifestyle-related background factor that was associated with readmission (p=0.017). Based on these findings, supporting patients with ALD in maintaining regular eating habits and taking experiences of loss into consideration would be important in avoiding readmission over the short-term.

U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

Translation initiation mediated by RNA looping.

Eukaryotic translation initiation commences at the initiation codon near the 5' end of mRNA by a 40S ribosomal subunit, and the recruitment of a 40S ribosome to an mRNA is facilitated by translation initiation factors interacting with the m(7)G cap and/or poly(A) tail. The 40S ribosome recruited to an mRNA is then transferred to the AUG initiation codon with the help of translation initiation factors. To understand the mechanism by which the ribosome finds an initiation codon, we investigated the role of eIF4G in finding the translational initiation codon. An artificial polypeptide eIF4G fused with MS2 was localized downstream of the reporter gene through MS2-binding sites inserted in the 3' UTR of the mRNA. Translation of the reporter was greatly enhanced by the eIF4G-MS2 fusion protein regardless of the presence of a cap structure. Moreover, eIF4G-MS2 tethered at the 3' UTR enhanced translation of the second cistron of a dicistronic mRNA. The encephalomyocarditis virus internal ribosome entry site, a natural translational-enhancing element facilitating translation through an interaction with eIF4G, positioned downstream of a reporter gene, also enhanced translation of the upstream gene in a cap-independent manner. Finally, we mathematically modeled the effect of distance between the cap structure and initiation codon on the translation efficiency of mRNAs. The most plausible explanation for translational enhancement by the translational-enhancing sites is recognition of the initiation codon by the ribosome bound to the ribosome-recruiting sites through "RNA looping." The RNA looping hypothesis provides a logical explanation for augmentation of translation by enhancing elements located upstream and/or downstream of a protein-coding region.

A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype.

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in, and the mechanistic basis of, CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF), p15(INK4B), and p16(INK4A), encoded by the INK4-ARF locus. In this study, we perform an RNA interference screen and identify ZNF304, a zinc-finger DNA-binding protein, as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors, ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1, resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304, which drives DNA binding. Finally, we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001.

Resistance to vemurafenib resulting from a novel mutation in the BRAFV600E kinase domain.

Resistance to the BRAF inhibitor vemurafenib poses a significant problem for the treatment of BRAFV600E-positive melanomas. It is therefore critical to prospectively identify all vemurafenib resistance mechanisms prior to their emergence in the clinic. The vemurafenib resistance mechanisms described to date do not result from secondary mutations within BRAFV600E. To search for possible mutations within BRAFV600E that can confer drug resistance, we developed a systematic experimental approach involving targeted saturation mutagenesis, selection of drug-resistant variants, and deep sequencing. We identified a single nucleotide substitution (T1514A, encoding L505H) that greatly increased drug resistance in cultured cells and mouse xenografts. The kinase activity of BRAFV600E/L505H was higher than that of BRAFV600E, resulting in cross-resistance to a MEK inhibitor. However, BRAFV600E/L505H was less resistant to several other BRAF inhibitors whose binding sites were further from L505 than that of PLX4720. Our results identify a novel vemurafenib-resistant mutant and provide insights into the treatment for melanomas bearing this mutation.

Fluorescent peptide indicator displacement assay for monitoring interactions between RNA and RNA binding proteins.

This paper describes a sensitive, non-destructive displacement assay, using a fluorescent peptide indicator, for real-time monitoring of the interactions between RNA and RNA binding proteins (RBPs). The developed fluorescent peptide indicators, each containing a mid-sequence fluorophore unit, allowed sensing of target RNA and RNA-RBP interactions through changes in fluorescence intensity. We anticipate that this assay will open up new possibilities for meaningful studies of RNA-RBP interactions.

The specific infectivity of hepatitis C virus changes through its life cycle.

Hepatitis C virus (HCV) causes liver diseases, such as hepatitis, liver cirrhosis, steatosis, and hepatocellular carcinoma. To understand the life cycle and pathogenesis of HCV, the one-step growth of HCV in a cell culture system was analyzed using a highly infectious variant of the JFH1 clone. The observed profiles of HCV RNA replication indicated that the synthesis of negative-strand RNAs occurred at 6 h (h) after infection, followed by the active synthesis of positive-strand RNAs. Our measurements of infectious virus production showed that the latent period of HCV was about 12 h. The specific infectivity of HCV particles (focus-forming unit per viral RNA molecule) secreted to the extracellular milieu early in infection was about 30-fold higher than that secreted later during infection. The buoyant densities of the infectious virion particles differed with the duration of infection, indicating changes in the compositions of the virion particles.

Cap-dependent translation without base-by-base scanning of an messenger ribonucleic acid.

'Ribosome scanning' is the generally accepted mechanism for explaining how a ribosome finds an initiation codon located far removed from the ribosome recruiting site (cap structure). However, the molecular characteristics of ribosome scanning along 5' untranslated regions (UTRs) remain obscure. Herein, using a rabbit reticulocyte lysate (RRL) system and artificial ribonucleic acid (RNA) constructs composed of a capped leader RNA and an uncapped reporter RNA annealed through a double-stranded RNA (dsRNA) bridge, we show that the ribosome can efficiently bypass a stable, dsRNA region without melting the structure. The insertion of an upstream open reading frame in the capped leader RNA impaired the translation of reporter RNA, indicating that a ribosome associated with the 5'-end explores the regions upstream of the dsRNA bridge in search of the initiation codon. These data indicate that a ribosome may skip part(s) of an messenger RNA 5'UTR without thoroughly scanning it.

Two cases of high output heart failure caused by hereditary hemorrhagic telangiectasia.

High-output cardiac failure is a rare complication of hereditary hemorrhagic telangiectasia (HHT) usually caused by shunting of blood through atriovenous malformations (AVMs) in the liver. We describe two cases of high output heart failure due to large hepatic AVMs. Clinical suspicion of HHT based on detailed history taking and physical examination is essential for early detection and proper management of heart failure associated with HHT.

A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53-) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

Rhythmic interaction between Period1 mRNA and hnRNP Q leads to circadian time-dependent translation.

The mouse PERIOD1 (mPER1) protein, along with other clock proteins, plays a crucial role in the maintenance of circadian rhythms. mPER1 also provides an important link between the circadian system and the cell cycle system. Here we show that the circadian expression of mPER1 is regulated by rhythmic translational control of mPer1 mRNA together with transcriptional modulation. This time-dependent translation was controlled by an internal ribosomal entry site (IRES) element in the 5' untranslated region (5'-UTR) of mPer1 mRNA along with the trans-acting factor mouse heterogeneous nuclear ribonucleoprotein Q (mhnRNP Q). Knockdown of mhnRNP Q caused a decrease in mPER1 levels and a slight delay in mPER1 expression without changing mRNA levels. The rate of IRES-mediated translation exhibits phase-dependent characteristics through rhythmic interactions between mPer1 mRNA and mhnRNP Q. Here, we demonstrate 5'-UTR-mediated rhythmic mPer1 translation and provide evidence for posttranscriptional regulation of the circadian rhythmicity of core clock genes.

Translation-competent 48S complex formation on HCV IRES requires the RNA-binding protein NSAP1.

Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).

eIF2A mediates translation of hepatitis C viral mRNA under stress conditions.

Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.

The novel gene twenty-four defines a critical translational step in the Drosophila clock.

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that show weak behavioural rhythms. Weak rhythms are accompanied by marked reductions in the levels of the clock protein Period (PER) as well as more modest effects on Timeless (TIM). Nonetheless, PER induction in pacemaker neurons can rescue tyf mutant rhythms. TYF associates with a 5'-cap-binding complex, poly(A)-binding protein (PABP), as well as per and tim transcripts. Furthermore, TYF activates reporter expression when tethered to reporter messenger RNA even in vitro. Taken together, these data indicate that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock.

RNA-binding protein hnRNP D modulates internal ribosome entry site-dependent translation of hepatitis C virus RNA.

Hepatitis C virus (HCV) is one of the major causative agents of virus-related hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) in the 5' nontranslated region of the genome. Here, we report that a cellular protein, hnRNP D, interacts with the 5' border of HCV IRES (stem-loop II) and promotes translation of HCV mRNA. Overexpression of hnRNP D in mammalian cells enhances HCV IRES-dependent translation, whereas knockdown of hnRNP D with small interfering RNAs (siRNAs) inhibits translation. In addition, sequestration of hnRNP D with an interacting DNA oligomer inhibits the translation of HCV mRNA in an in vitro system. Ribosome profiling experiments reveal that HCV RNA is redistributed from heavy to light polysome fractions upon suppression of the hnRNP D level using specific siRNA. These results collectively suggest that hnRNP D plays an important role in the translation of HCV mRNA through interactions with the IRES. Moreover, knockdown of hnRNP D with siRNA significantly hampers infection by HCV. A potential role of hnRNP D in HCV proliferation is discussed.

Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

BiP internal ribosomal entry site activity is controlled by heat-induced interaction of NSAP1.

TheBiP protein, a stress response protein, plays an important role in the proper folding and assembly of nascent protein and in the scavenging of misfolded proteins in the endoplasmic reticulum lumen. Translation of BiP is directed by an internal ribosomal entry site (IRES) in the 5' nontranslated region of the BiP mRNA. BiP IRES activity increases when cells are heat stressed. Here we report that NSAP1 specifically enhances the IRES activity of BiP mRNA by interacting with the IRES element. Overexpression of NSAP1 in 293T cells increased the IRES activity of BiP mRNA, whereas knockdown of NSAP1 by small interfering RNA (siRNA) reduced the IRES activity of BiP mRNA. The amount of NSAP1 bound to the BiP IRES increased under heat stress conditions, and the IRES activity of BiP mRNA was increased. Moreover, the increase in BiP IRES activity with heat treatment was not observed in cells lacking NSAP1 after siRNA treatment. BiP mRNAs were redistributed from the heavy polysome to the light polysome in NSAP1 knockdown cells. Together, these data indicate that NSAP1 modulates IRES-dependent translation of BiP mRNA through an RNA-protein interaction under heat stress conditions.

Efficacy of irbesartan on left ventricular mass and arterial stiffness in hypertensive patients.

BACKGROUND: Increased aortic stiffness measured by pulse wave velocity (PWV) and left ventricular hypertrophy (LVH) are independent risk factors of cardiovascular events in hypertensive patients. We have conducted a prospective study to examine the effects of the angiotensin II receptor antagonist (irbesartan) on PWV and LVH in hypertensive patients. METHODS: A total of 52 untreated hypertensive patients (age: 53.3 +/- 8.0 yrs) were enrolled; they had no evidence of associated cardiovascular complications. Blood pressure, heart rate, aortic PWV and left ventricular mass index (LVMI) by 2-D echocardiography were measured at baseline and after irbesartan treatment (150 mg or 300 mg/day) at 12 weeks and 24 weeks. RESULTS: Blood pressure was significantly decreased after 12 weeks and 24 weeks of treatment compared to baseline (SBP: 134.6 +/- 13.3 mmHg, 134.0 +/- 11.0 mmHg vs 163.7 +/- 13.8 mmHg p < 0.001, DBP: 86.0 +/- 10 mmHg, 83.07 mmHg vs 102.4 +/- 9.6 mmHg p < 0.001, respectively) without significant change in heart rate. LVMI decreased at 12 weeks and at 24 weeks after treatment compared to baseline (from 145.5 +/- 35.1 g/m2 at baseline to 137.5 +/- 35.4 g/m2 at 12 weeks, p = 0.017 and 135.3 +/- 35.4 g/m2 at 24 weeks, p = 0.008). Aortic PWV was decreased after irbesartan treatment at 12 weeks (from 9.6 +/- 2.8 m/sec to 8.7 +/- 3.1 m/sec at 12 weeks, p = 0.064) and at 24 weeks (from 9.6 +/- 2.9 m/sec to 7.7 +/- 2.1 m/sec at 24 weeks, p = 0.007). CONCLUSIONS: Long-term treatment with irbesartan may reduce arterial stiffness and regression of LVH in hypertensive patients. The pleiotropic effects of irbesartan, further decreasing PWV without change of BP between 12 and 24 weeks of treatment, may have favorable vascular effects on arterial stiffness and LVH.