Bicistronic Transcript Research Articles

Upstream open reading frames may contain hundreds of novel human exons.

Several recent studies have presented evidence that the human gene catalogue should be expanded to include thousands of short open reading frames (ORFs) appearing upstream or downstream of existing protein-coding genes, each of which might create an additional bicistronic transcript in humans. Here we explore an alternative hypothesis that would explain the translational and evolutionary evidence for these upstream ORFs without the need to create novel genes or bicistronic transcripts. We examined 2,199 upstream ORFs that have been proposed as high-quality candidates for novel genes, to determine if they could instead represent protein-coding exons that can be added to existing genes. We checked for the conservation of these ORFs in four recently sequenced, high-quality human genomes, and found a large majority (87.8%) to be conserved in all four as expected. We then looked for splicing evidence that would connect each upstream ORF to the downstream protein-coding gene at the same locus, thus creating a novel splicing variant using the upstream ORF as its first exon. These protein coding exon candidates were further evaluated using protein structure predictions of the protein sequences that included the proposed new exons. We determined that 541 out of 2,199 upstream ORFs have strong evidence that they can form protein coding exons that are part of an existing gene, and that the resulting protein is predicted to have similar or better structural quality than the currently annotated isoform.

Staphylococcus aureus SigS Induces Expression of a Regulatory Protein Pair That Modulates Its mRNA Stability.

SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.

Generation of a novel Stra8-driven Cre recombinase strain for use in pre-meiotic germ cells in mice†.

The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility.

Improved gene therapy for MFRP deficiency-mediated retinal degeneration by knocking down endogenous bicistronic Mfrp and Ctrp5 transcript

The membrane frizzled-related protein (Mfrp) and C1-tumor necrosis factor related protein 5 (Ctrp5) genes are transcribed as a bicistronic unit and dysregulation of either gene is associated with retinal degeneration in the retinal pigment epithelium (RPE) cells. However, the mechanisms that regulate the expression of the bicistronic transcript remain controversial. Here, we identified a microRNA-based negative feedback loop that helps maintain a normal expression level of the bicistronic Mfrp and Ctrp5 transcript. Specifically, miR-149-3p, a conserved microRNA, binds to the 3'UTR of the Mfrp gene. In MFRP-deficient rd6 mice, the miR-149-3p levels were compromised compared with those in WT mice, resulting in an increase in the bicistronic transcript. We also report a capsid-modified rAAVDJ-3M vector that is capable of robustly and specifically transducing RPE cells following subretinal delivery. Compared with the parental vector, the modified vector elicited similar levels of serum anti-rAAV antibodies, but recruited fewer microglial infiltrations. Most significantly, we also demonstrate that simultaneous overexpressing of MFRP and knockdown of the bicistronic transcript was more effective in rescuing vision than MFRP overexpression alone. Our findings offer new insights into the function of MFRP and provide a promising therapeutic strategy for the treatment of MFRP-associated ocular diseases.

A Small Non-Coding RNA Mediates Transcript Stability and Expression of Cytochrome bd Ubiquinol Oxidase Subunit I in Rickettsia conorii.

Small regulatory RNAs (sRNAs) are now widely recognized for their role in the post-transcriptional regulation of bacterial virulence and growth. We have previously demonstrated the biogenesis and differential expression of several sRNAs in Rickettsia conorii during interactions with the human host and arthropod vector, as well as the in vitro binding of Rickettsia conorii sRNA Rc_sR42 to bicistronic cytochrome bd ubiquinol oxidase subunits I and II (cydAB) mRNA. However, the mechanism of regulation and the effect of sRNA binding on the stability of the cydAB bicistronic transcript and the expression of the cydA and cydB genes are still unknown. In this study, we determined the expression dynamics of Rc_sR42 and its cognate target genes, cydA and cydB, in mouse lung and brain tissues during R. conorii infection in vivo and employed fluorescent and reporter assays to decode the role of sRNA in regulating cognate gene transcripts. Quantitative RT-PCR revealed significant changes in the expression of sRNA and its cognate target gene transcripts during R. conorii infection in vivo, and a greater abundance of these transcripts was observed in the lungs compared to brain tissue. Interestingly, while Rc_sR42 and cydA exhibited similar patterns of change in their expression, indicating the influence of sRNA on the mRNA target, the expression of cydB was independent of sRNA expression. Further, we constructed reporter plasmids of sRNA and cydAB bicistronic mRNA to decipher the role of sRNA on CydA and CydB expression. We observed increased expression of CydA in the presence of sRNA but detected no change in CydB expression in the presence or absence of sRNA. In sum, our results demonstrate that the binding of Rc_sR42 is required for the regulation of cydA but not cydB. Further studies on understanding the influence of this interaction on the mammalian host and tick vector during R. conorii infection are in progress.

Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in Drosophila.

Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open-reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.

Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitoribosomal RNA of the minor subunit, 15S rRNA, is transcribed as a bicistronic transcript along with tRNAW. 5' and 3' sequences flanking the mature transcript must be removed by cleavage at the respective junctions before incorporating it into the mitoribosome. An in vivo dose-response triphasic system was created to elucidate the role of Ccm1p in the processing of 15S rRNA: Ccm1p supply ("On"), deprivation ("Off"), and resupply ("Back on"). After 72h under "Off" status, the cells started to exhibit a complete mutant phenotype as assessed by their lack of growth in glycerol medium, while keeping their mitochondrial DNA integrity (ρ+). Full functionality of mitochondria was reacquired upon "Back on." 15S rRNA levels and phenotype followed the Ccm1p intramitochondrial concentrations throughout the "On-Off-Back on" course. Under "Off" status, cells gradually accumulated unprocessed 5' and 3' junctions, which reached significant levels at 72-96h, probably due to a saturation of the mitochondrial degradosome (mtEXO). The Ccm1p/mtEXO mutant (Δccm1/Δdss1) showed a copious accumulation of 15S rRNA primary transcript forms, which were cleaved upon Ccm1p resupply. The gene that codes for the RNA component of RNase P was conserved in wild-type and mutant strains. Our results indicate that Ccm1p is crucial in processing the 15S rRNA primary transcript and does not stabilize the already mature 15S rRNA. Consequently, failure of this function in Δccm1 cells results, as it happens to any other unprocessed primary transcripts, in total degradation of 15S rRNA by mtEXO, whose mechanism of action is discussed.

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly.

SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions.

GDF1 Regulation of Ceramide Metabolism Restores Effective Hematopoiesis in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by ineffective hematopoiesis, cytopenias, and an increased risk of transformation to acute myeloid leukemia. New studies are urgently needed to understand the underlying processes that govern MDS and related bone marrow failure disorders and to develop better therapeutic modalities. Sphingolipids are an extensive classification of lipids which play prominent roles in cellular signaling in addition to being essential components of membranes. The most well studied sphingolipid is ceramide, which serves as a hypothetical center of sphingolipid metabolism. Ceramide is an important cellular signal that can lead to apoptosis. Overall ceramide biology is significant because novel metabolic routes that persist in MDS may be exploited for therapeutic development. Interestingly, the gene encoding ceramide synthase 1 is encoded from a bicistronic transcript that also encodes for GDF1. Little is known about the roles of GDF1 outside of cardiac development. However, GDF1 is in the TGF-beta superfamily of which many members have been attributed roles in stem cell biology. Therefore, a better understanding of the role of GDF1 and its regulation of ceramide metabolism and hematopoiesis may lead to better treatment approaches for MDS and other bone marrow failure syndromes. The current study tested an overarching hypothesis that GDF1 regulates SMAD and STAT signaling to promote ceramide generation and restore effective myelopoiesis in MDS. Initially, the expression of GDF1 was evaluated in hematopoietic cells isolated from transgenic murine models of MDS (Nup98-HoxD13; Srsf2P95H-mutant) as well as myeloproliferation (Flt3ITD). Interestingly, GDF1 expression was greatest in the bone marrow of MDS models. This was significant because we recently reported that nanoliposomal ceramide (Lip-C6), which delivers a bioactive ceramide analog, exerts unique therapeutic efficacy towards acute myeloid leukemia arising out of MDS (Barth et. al. Blood Advances 2019). Lip-C6 is a ceramide-based therapy that currently is in a clinical trial for solid tumor malignancies (ClinicalTrials.gov identifier: NCT02834611). Next, treatment of transgenic MDS mice with either recombinant GDF1 or Lip-C6 was shown to stimulate the expansion of erythroid progenitors. This was concomitant with a decrease in immature myeloid cells, which was revealed to be due to granulo-monocytopoietic differentiation. Mechanistic studies subsequently revealed that GDF1 increased SMAD2/3 phosphorylation while simultaneously down regulating STAT3 (Y705) phosphorylation, both in a TGF-beta receptor 1-dependent fashion. This dual effect was unique to GDF1, whereas TGF-beta or GDF3 only recapitulated individual the effects on SMAD2/3 and STAT3 signaling, respectively. Finally, STAT3 binding sites were identified in the promoter region of the ceramide detoxifying enzyme glucosylceramide synthase. This is important because glucosylceramide synthase expression was shown to be downregulated by recombinant GDF1 treatment. Therefore, GDF1-mediated downregulation of STAT3-dependent glucosylceramide synthase expression provides a mechanistic link where GDF1 can augment intracellular ceramide levels. Overall, this study enhances our understanding of a fundamental hematopoietic process where GDF1 regulates ceramide metabolism and myelopoiesis. This is impactful because the effects of GDF1, which are unique from other related factors including TGF-beta and GDF3, are due to a novel mechanism of action that both upregulates SMAD2/3 signaling while downregulating STAT3 signaling. More so, this study demonstrated that GDF1, and/or ceramide, can exert an anti-MDS therapeutic role by restoring normal aspects of myelopoiesis. This work was funded by NIH/NCI K22 CA190674 (B.M.B.) and University of New Hampshire COBRE Pilot Project Grant NIH/NIGMS P20 GM113131 (B.M.B.). The authors acknowledge US Provisional Patent 62/602,437, issued to B.M.B. and the University of New Hampshire. Disclosures Barth: University of New Hampshire: Patents &amp; Royalties: US Provisional Patent 62/602,437; NIH (NCI and NIGMS): Research Funding. Loughran:Bioniz: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees.

Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.

GaHV-2 ICP22 protein is expressed from a bicistronic transcript regulated by three GaHV-2 microRNAs.

Herpesviruses have a lifecycle consisting of successive lytic, latent and reactivation phases. Only three infected cell proteins (ICPs) have been described for the oncogenic Marek's disease virus (or Gallid herpes virus 2, GaHV-2): ICP4, ICP22 and ICP27. We focus here on ICP22, confirming its cytoplasmic location and showing that ICP22 is expressed during productive phases of the lifecycle, via a bicistronic transcript encompassing the US10 gene. We also identified the unique promoter controlling ICP22 expression, and its core promoter, containing functional responsive elements including E-box, ETS-1 and GATA elements involved in ICP22 transactivation. ICP22 gene expression was weakly regulated by DNA methylation and activated by ICP4 or ICP27 proteins. We also investigated the function of GaHV-2 ICP22. We found that this protein repressed transcription from its own promoter and from those of IE ICP4 and ICP27, and the late gK promoter. Finally, we investigated posttranscriptional ICP22 regulation by GaHV-2 microRNAs. We found that mdv1-miR-M5-3p and -M1-5p downregulated ICP22 mRNA expression during latency, whereas, unexpectedly, mdv1-miR-M4-5p upregulated the expression of the protein ICP22, indicating a tight regulation of ICP22 expression by microRNAs.

C33-A cells transfected with E6*I or E6*II the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis

The HPV-16 E6/E7 bicistronic immature transcript produces 4 mature RNAs: the unspliced HPV-16 E6/E7pre−mRNA product and 3 alternatively spliced mRNAs. The 3 spliced mRNAs encode short forms of the E6 oncoprotein, namely E6*I, E6*II and E6^E7. In this study we showed that transfection of C-33A cells with monocistronic constructs of these cDNAs fused to GFP, produced different effects on apoptosis, after the treatment with cisplatin. Transfection of C-33A cells with the full-length E6-GFP oncoprotein resulted in a 50% decrease in cell death, while the transfection with the E6*I-GFP construct showed only a 25% of diminution of cell death, compared to the control cells. Transfection with the E6^E7-GFP or E7-GFP construct had no effect on the number of the apoptotic cells, compared with control cells. Conversely, transfection with the E6*II construct resulted in higher cell death than the control cells. Taken together, these results suggested that E6*I or E6*II, the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis, when transfected in C-33A cells.

A Natural Vibrio parahaemolyticus ΔpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

ABSTRACTAcute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirAVp and PirBVp. These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirAVp but carried pirBVp. Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirBVp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirAVp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirAVp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirAVp gene sequence and in the downstream pirBVp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirABVp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no PirVp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirAVp and PirBVp. The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND. Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND. Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

P2A-Fluorophore Tagging of BRAF Tightly Links Expression to Fluorescence In Vivo.

The Braf proto-oncogene is a key component of the mitogen-activated protein kinase signaling cascade and is a critical regulator of both normal development and tumorigenesis in a variety of tissues. In order to elucidate BRAF’s differing roles in varying cell types, it is important to understand both the pattern and timing of BRAF expression. Here we report the production of a mouse model that links the expression of Braf with the bright red fluorescent protein, tdTomato. We have utilized a P2A knock-in strategy, ensuring that BRAF and the fluorophore are expressed from the same endogenous promoter and from the same bicistronic mRNA transcript. This mouse model (BrafTOM) shows bright red fluorescence in organs and cell types known to be sensitive to BRAF perturbation. We further show that on a cell-by-cell basis, fluorescence correlates with BRAF protein levels. Finally, we extend the utility of this mouse by demonstrating that the remnant P2A fragment attached to BRAF acts as a suitable epitope for immunoprecipitation and biochemical characterization of BRAF in vivo.

Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system.

Restriction–modification (R-M) systems are highly prevalent among bacteria and archaea, and appear to play crucial roles in modulating horizontal gene transfer and protection against phage. There is much to learn about these diverse enzymes systems, especially their regulation. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). Their activities need to be finely balanced in vivo. Some R-M systems rely on specialized transcription factors called C (controller) proteins. These proteins play a vital role in the temporal regulation of R-M gene expression, and function to indirectly modulate the horizontal transfer of their genes across the species. We report novel regulation of a C-responsive R-M system that involves a C protein of a poorly-studied structural class - C.Csp231I. Here, the C and REase genes share a bicistronic transcript, and some of the transcriptional auto-control features seen in other C-regulated R-M systems are conserved. However, separate tandem promoters drive most transcription of the REase gene, a distinctive property not seen in other tested C-linked R-M systems. Further, C protein only partially controls REase expression, yet plays a role in system stability and propagation. Consequently, high REase activity was observed after deletion of the entire C gene, and cells bearing the ΔC R-M system were outcompeted in mixed culture assays by those with the WT R-M system. Overall, our data reveal unexpected regulatory variation among R-M systems.

A Bicistronic MAVS Transcript Highlights a Class of Truncated Variants in Antiviral Immunity

Bacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full-length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA and suggests a widespread role for polycistronic mRNAs in the innate immune system.

LDL-receptor-deficient mice lacking microRNA-143/145 have less atherosclerosis.

MicroRNAs (miRNAs, miRs) have emerged as important post-transcriptional regulators of gene expression. In vascular pathologies, the miR-143/145 cluster has attracted particular attention. The miR-143/145 cluster is encoded by a bicistronic transcript, and regulates the differentiation, plasticity and contractile function of vascular smooth muscle cells (VSMCs). VSMCs from miR143/145–deficient mice were locked in a synthetic state, which favoured neointimal lesion development despite normal levels of serum cholesterol and lipoproteins (1). In contrast, deficiency of miR-143/145 on the low-density lipoprotein receptor (Ldlr-/-) background reduced VLDL and LDL cholesterol (2). When Sala et al. crossed miR-143/145-/mice with Ldlr-/mice, the double knock-out mice had less atherosclerosis. Bioinformatic prediction algorithms identified the ATP-binding cassette transporter ABCA1 as a potential target of miR-145. Functional and luciferase experiments confirmed a direct interaction of miR-145 with the 3’UTR of ABCA1. In tissues from Ldlr-/mice, miR-145 levels were significantly higher in the aorta than in liver or macrophages. In the double knockout mice, ABCA1 expression was markedly increased in the aorta and the liver, but there was no change in HDL cholesterol although ABCA1 promotes cellular cholesterol efflux. The macrophage content in atherosclerotic plaques, however, was decreased. Based on an in vitro approach, the authors postulated that miR-145 may be transferred from VSMCs to macrophages. A similar transfer of miR-143/145 had previously been reported for endothelial cells: miR-143/145 secreted in exosomes of endothelial cells could apparently regulate VSMC function (3). It is currently unclear whether such a miRNA transfer between vascular cell types is just an in vitro phenomenon or also occurs in vivo to modify gene expression. In summary, therapeutic strategies targeting miR-145 to maintain a contractile VSMC phenotype have to be reevaluated in the context of dyslipidaemia.

Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

Analysis of the Organic Hydroperoxide Response of Chromobacterium violaceum Reveals That OhrR Is a Cys-Based Redox Sensor Regulated by Thioredoxin

Organic hydroperoxides are oxidants generated during bacterial-host interactions. Here, we demonstrate that the peroxidase OhrA and its negative regulator OhrR comprise a major pathway for sensing and detoxifying organic hydroperoxides in the opportunistic pathogen Chromobacterium violaceum. Initially, we found that an ohrA mutant was hypersensitive to organic hydroperoxides and that it displayed a low efficiency for decomposing these molecules. Expression of ohrA and ohrR was specifically induced by organic hydroperoxides. These genes were expressed as monocistronic transcripts and also as a bicistronic ohrR-ohrA mRNA, generating the abundantly detected ohrA mRNA and the barely detected ohrR transcript. The bicistronic transcript appears to be processed. OhrR repressed both the ohrA and ohrR genes by binding directly to inverted repeat sequences within their promoters in a redox-dependent manner. Site-directed mutagenesis of each of the four OhrR cysteine residues indicated that the conserved Cys21 is critical to organic hydroperoxide sensing, whereas Cys126 is required for disulfide bond formation. Taken together, these phenotypic, genetic and biochemical data indicate that the response of C. violaceum to organic hydroperoxides is mediated by OhrA and OhrR. Finally, we demonstrated that oxidized OhrR, inactivated by intermolecular disulfide bond formation, is specifically regenerated via thiol-disulfide exchange by thioredoxin (but not other thiol reducing agents such as glutaredoxin, glutathione and lipoamide), providing a physiological reducing system for this thiol-based redox switch.

Transcription of gD and gI genes in BHV1-infected cells

Glycoprotein D (gD) and glycoprotein I (gI) genes of bovine herpes virus 1 (BHV1) are contiguous genes with 141 bp region between the two open reading frames (ORFs). Expression of gD and gI from a bicistronic construct containing complete gD and gI gene has been reported either through internal ribosome entry site (IRES)-like element or through the scanning and leakage model (Mukhopadhyay 2000). We here show by computational and experimental means that gD is expressed solely as bicistronic transcript comprising gD and gI coding region in BHV1-infected cells. gI ORF was also shown to express separately. An IRES-like element was also predicted by IRES predicting software in the middle of the gD coding region; within that region a putative promoter was also identified by promoterscan. The intergenic region between the two ORF showed extensive secondary structure which brings the stop codon of gD very close to start codon of gI gene. gD gene transcript in BHV1-infected cells was solely bicistronic. gI transcript was also present in the BHV1-infected cells but in low copy number. The results indicate that gI is probably transcribed from its own transcript in BHV1-infected cells.