Comt1 genotype and expression predicts anxiety and nociceptive sensitivity in inbred strains of mice

Catechol‐O‐methyltransferase (COMT) is a ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity and risk for developing psychiatric disorders. A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure‐dependent protein translation. However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have assessed Comt1 gene expression levels in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed among the strains, and this differential expression is cis‐regulated. A B2 short interspersed nuclear element (SINE) was inserted in the 3′‐untranslated region (3′‐UTR) of Comt1 in 14 strains generating a common haplotype that correlates with gene expression. Experiments using mammalian expression vectors of full‐length cDNA clones with and without the SINE element show that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3′‐UTR B2 SINE element, regulates Comt1 expression and some mouse behaviors.     

Allele Specific Expression of the Transthyretin Gene in Swedish Patients with Hereditary Transthyretin Amyloidosis (ATTR V30M) Is Similar between the Two Alleles

Background

Hereditary transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease characterized by extracellular deposits of amyloid fibrils composed of misfolded TTR. The differences in penetrance and age at onset are vast, both between and within populations, with a generally late onset for Swedish carriers. In a recent study the entire TTR gene including the 3′ UTR in Swedish, French and Japanese ATTR patients was sequenced. The study disclosed a SNP in the V30M TTR 3′ UTR of the Swedish ATTR population that was not present in either the French or the Japanese populations (rs62093482-C>T). This SNP could create a new binding site for miRNA, which would increase degradation of the mutated TTR’s mRNA thus decrease variant TTR formation and thereby delay the onset of the disease. The aim of the present study was to disclose differences in allele specific TTR expression among Swedish V30M patients, and to see if selected miRNA had any effect upon the expression.

Methodology/Principal Findings

Allele-specific expression was measured on nine liver biopsies from Swedish ATTR patients using SNaPshot Multiplex assay. Luciferase activity was measured on cell lines transfected with constructs containing the TTR 3′ UTR. Allele-specific expression measured on liver biopsies from Swedish ATTR patients showed no difference in expression between the two alleles. Neither was there any difference in expression between cell lines co-transfected with two constructs with or without the TTR 3′ UTR SNP regardless of added miRNA.

Conclusions/Significance

The SNP found in the 3′ UTR of the TTR gene has no effect on degrading the variant allele’s expression and thus has no impact on the diminished penetrance of the trait in the Swedish population. However, the 3′ UTR SNP is unique for patients descending from the Swedish founder, and this SNP could be utilized to identify ATTR patients of Swedish descent.     

Tumor suppression by RNA from C/EBPβ 3'UTR through the inhibition of protein kinase Cε activity

Background

Since the end of last century, RNAs from the 3′untranslated region (3′UTR) of several eukaryotic mRNAs have been found to exert tumor suppression activity when introduced into malignant cells independent of their whole mRNAs. In this study, we sought to determine the molecular mechanism of the tumor suppression activity of a short RNA from 3′UTR of C/EBPβ mRΝΑ (C/EBPβ 3′UTR RNA) in human hepatocarcinoma cells SMMC-7721.

Methodology/Principal Findings

By using Western blotting, immunocytochemistry, molecular beacon, confocal microscopy, protein kinase inhibitors and in vitro kinase assays, we found that, in the C/EBPβ 3′UTR-transfectant cells of SMMC-7721, the overexpressed C/EBPβ 3′UTR RNA induced reorganization of keratin 18 by binding to this keratin; that the C/EBPβ 3′UTR RNA also reduced phosphorylation and expression of keratin 18; and that the enzyme responsible for phosphorylating keratin 18 is protein kinase Cε. We then found that the C/EBPβ 3′UTR RNA directly inhibited the phosphorylating activity of protein kinase Cε; and that C/EBPβ 3′UTR RNA specifically bound with the protein kinase Cε-keratin 18 conjugate.

Conclusion/Significance

Together, these facts suggest that the tumor suppression in SMMC-7721 by C/EBPβ 3′UTR RNA is due to the inhibition of protein kinase Cε activity through direct physical interaction between C/EBPβ 3′UTR RNA and protein kinase Cε. These facts indicate that the 3′UTR of some eukaryotic mRNAs may function as regulators for genes other than their own.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.     

Targeted delivery of mutant tolerant anti-coxsackievirus artificial microRNAs using folate conjugated bacteriophage Phi29 pRNA

Background

Myocarditis is the major heart disease in infants and young adults. It is very commonly caused by coxsackievirus B3 (CVB3) infection; however, no specific treatment or vaccine is available at present. RNA interference (RNAi)-based anti-viral therapy has shown potential to inhibit viral replication, but this strategy faces two major challenges; viral mutational escape from drug suppression and targeted delivery of the reagents to specific cell populations.

Methodology/Principal Findings

In this study, we designed artificial microRNAs (AmiRs) targeting the 3′untranslated region (3′UTR) of CVB3 genome with mismatches to the central region of their targeting sites. Antiviral evaluation showed that AmiR-1 and AmiR-2 reduced CVB3 (Kandolf and CG strains) replication approximately 100-fold in both HeLa cells and HL-1 cardiomyoctes. To achieve specific delivery, we linked AmiRs to the folate-conjugated bacterial phage packaging RNA (pRNA) and delivered the complexes into HeLa cells, a folate receptor positive cancer cells widely used as an in vitro model for CVB3 infection, via folate-mediated specific internalization. We found that our designed pRNA-AmiRs conjugates were tolerable to target mutations and have great potential to suppress viral mutational escape with little effect on triggering interferon induction.

Conclusion/Significance

This study provides important clues for designing AmiRs targeting the 3′UTR of viral genome. It also proves the feasibility of specific deliver of AmiRs using conjugated pRNA vehicles. These small AmiRs combined with pRNA-folate conjugates could form a promising system for antiviral drug development.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

Evidence of Recombination and Genetic Diversity in Human Rhinoviruses in Children with Acute Respiratory Infection

Background

Human rhinoviruses (HRVs) are a highly prevalent cause of acute respiratory infection in children. They are classified into at least three species, HRV-A, HRV-B and HRV-C, which are characterized by sequencing the 5′ untranslated region (UTR) or the VP4/VP2 region of the genome. Given the increased interest for novel HRV strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of HRV strains in a 2-year study of children with acute respiratory infection visiting one district hospital in Shanghai.

Methodology/Findings

We cloned and sequenced a 924-nt fragment that covered part of the 5′UTR and the VP4/VP2 capsid genes. Sixty-four HRV-infected outpatients were diagnosed amongst 827 children with acute low respiratory tract infection. Two samples were co-infected with HRV-A and HRV-B or HRV-C. By comparative analysis of the VP4/VP2 sequences of the 66 HRVs, we showed a high diversity of strains in HRV-A and HRV-B species, and a prevalence of 51.5% of strains that belonged to the recently identified HRV-C species. When analyzing a fragment of the 5′ UTR, we characterized at least two subspecies of HRV-C: HRV-Cc, which clustered differently from HRV-A and HRV-B, and HRV-Ca, which resulted from previous recombination in this region with sequences related to HRV-A. The full-length sequence of one strain of each HRV-Ca and HRV-Cc subspecies was obtained for comparative analysis. We confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the 2A gene and 3′UTR of the HRV-Ca strain. Double or triple infections with HRV-C and respiratory syncytial virus and/or bocavirus were diagnosed in 33.3% of the HRV-infected patients, but no correlation with severity of clinical outcome was observed.

Conclusion

Our study showed a high diversity of HRV strains that cause bronchitis and pneumonia in children. A predominance of HRV-C over HRV-A and HRV-B was observed, and two subspecies of HRV-C were identified, the diversity of which seemed to be related to recombination with former HRV-A strains. None of the HRV-C strains appeared to have a higher clinical impact than HRV-A or HRV-B on respiratory compromise.     

The induction of microRNA targeting IRS-1 is involved in the development of insulin resistance under conditions of mitochondrial dysfunction in hepatocytes

Background

Mitochondrial dysfunction induces insulin resistance in myocytes via a reduction of insulin receptor substrate-1 (IRS-1) expression. However, the effect of mitochondrial dysfunction on insulin sensitivity is not understood well in hepatocytes. Although research has implicated the translational repression of target genes by endogenous non-coding microRNAs (miRNA) in the pathogenesis of various diseases, the identity and role of the miRNAs that are involved in the development of insulin resistance also remain largely unknown.

Methodology

To determine whether mitochondrial dysfunction induced by genetic or metabolic inhibition causes insulin resistance in hepatocytes, we analyzed the expression and insulin-stimulated phosphorylation of insulin signaling intermediates in SK-Hep1 hepatocytes. We used qRT-PCR to measure cellular levels of selected miRNAs that are thought to target IRS-1 3′ untranslated regions (3′UTR). Using overexpression of miR-126, we determined whether IRS-1-targeting miRNA causes insulin resistance in hepatocytes.

Principal Findings

Mitochondrial dysfunction resulting from genetic (mitochondrial DNA depletion) or metabolic inhibition (Rotenone or Antimycin A) induced insulin resistance in hepatocytes via a reduction in the expression of IRS-1 protein. In addition, we observed a significant up-regulation of several miRNAs presumed to target IRS-1 3′UTR in hepatocytes with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-126 directly targeted to IRS-1 3′UTR. Furthermore, the overexpression of miR-126 in hepatocytes caused a substantial reduction in IRS-1 protein expression, and a consequent impairment in insulin signaling.

Conclusions/Significance

We demonstrated that miR-126 was actively involved in the development of insulin resistance induced by mitochondrial dysfunction. These data provide novel insights into the molecular basis of insulin resistance, and implicate miRNA in the development of metabolic disease.     

Effect of β2-adrenergic receptor gene (ADRB2) 3′ untranslated region polymorphisms on inhaled corticosteroid/long-acting β2-adrenergic agonist response

Background

Evidence suggests that variation in the length of the poly-C repeat in the 3′ untranslated region (3′UTR) of the β₂-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in β-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the ADRB2 3′UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting β₂-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response.

Methods

In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed.

Results

Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients’ pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype.

Conclusions

The extensive sequence diversity present in the poly-C repeat region of the ADRB2 3′UTR did not predict therapeutic response to ICS/LABA therapy.     

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA

Background

Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation.

Methodology/Principal Findings

We have probed the putative secondary structure of the IRF2 5′UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3′terminus of the 5′untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2–5′UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3′end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA.

Conclusion/Significance

It appears that PTB binding to multiple sites within IRF2 5′UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation.     

MicroRNA-18a Attenuates DNA Damage Repair through Suppressing the Expression of Ataxia Telangiectasia Mutated in Colorectal Cancer

Background

miR-18a is one of the most up-regulated miRNAs in colorectal cancers (CRC) based on miRNA profiling. In this study, we examined the functional significance of miR-18a in CRC.

Methods

Expression of miR-18a was investigated in 45 CRC patients. Potential target genes of miR-18a were predicted by in silico search and confirmed by luciferase activity assay and Western blot. DNA damage was measured by comet assay. Gene function was measured by cell viability, colony formation and apoptosis assays.

Results

The up-regulation of miR-18a was validated and confirmed in 45 primary CRC tumors compared with adjacent normal tissues (p<0.0001). Through in silico search, the 3′UTR of Ataxia telangiectasia mutated (ATM) contains a conserved miR-18a binding site. Expression of ATM was down-regulated in CRC tumors (p<0.0001) and inversely correlated with miR-18a expression (r = -0.4562, p<0.01). Over-expression of miR-18a in colon cancer cells significantly reduced the luciferase activity of the construct with wild-type ATM 3′UTR but not that with mutant ATM 3′UTR, inferring a direct interaction of miR-18a with ATM 3′UTR. This was further confirmed by the down-regulation of ATM protein by miR-18a. As ATM is a key enzyme in DNA damage repair, we evaluated the effect of miR-18a on DNA double-strand breaks. Ectopic expression of miR-18a significantly inhibited the repair of DNA damage induced by etoposide (p<0.001), leading to accumulation of DNA damage, increase in cell apoptosis and poor clonogenic survival.

Conclusion

miR-18a attenuates cellular repair of DNA double-strand breaks by directly suppressing ATM, a key enzyme in DNA damage repair.