Polyadenylation Signal Research Articles

Ipa1 Is an RNA Polymerase II Elongation Factor that Facilitates Termination by Maintaining Levels of the Poly(A) Site Endonuclease Ysh1

SUMMARYThe yeast protein Ipa1 was recently discovered to interact with the Ysh1 endonuclease of the prem-RNA cleavage and polyadenylation (C/P) machinery, and Ipa1 mutation impairs 3′end processing. We report that Ipa1 globally promotes proper transcription termination and poly(A) site selection, but with variable effects on genes depending upon the specific configurations of polyadenylation signals. Our findings suggest that the role of Ipa1 in termination is mediated through interaction with Ysh1, since Ipa1 mutation leads to decrease in Ysh1 and poor recruitment of the C/P complex to a transcribed gene. The Ipa1 association with transcriptionally active chromatin resembles that of elongation factors, and the mutant shows defective Pol II elongation kinetics in vivo. Ysh1 overexpression in the Ipa1 mutant rescues the termination defect, but not the mutant’s sensitivity to 6-azauracil, an indicator of defective elongation. Our findings support a model in which an Ipa1/Ysh1 complex helps coordinate transcription elongation and 3′ end processing.

U1 snRNP Telescripting Roles in Transcription and Its Mechanism.

Telescripting is a fundamental cotranscriptional gene regulation process that relies on U1 snRNP (U1) to suppress premature 3'-end cleavage and polyadenylation (PCPA) in RNA polymerase II (Pol II) transcripts, which is necessary for full-length transcription of thousands of protein-coding (pre-mRNAs) and long noncoding (lncRNA) genes. Like U1 role in splicing, telescripting requires U1 snRNA base-pairing with nascent transcripts. Inhibition of U1 base-pairing with U1 snRNA antisense morpholino oligonucleotide (U1 AMO) mimics widespread PCPA from cryptic polyadenylation signals (PASs) in human tissues, including PCPA in introns and last exons' 3'-untranslated regions (3' UTRs). U1 telescripting-PCPA balance changes generate diverse RNAs depending on where in a gene it occurs. Long genes are highly U1-telescripting-dependent because of PASs in introns compared to short genes. Enrichment of cell cycle control, differentiation, and developmental functions in long genes, compared to housekeeping and acute cell stress response genes in short genes, reveals a gene size-function relationship in mammalian genomes. This polarization increased in metazoan evolution by previously unexplained intron expansion, suggesting that U1 telescripting could shift global gene expression priorities. We show that that modulating U1 availability can profoundly alter cell phenotype, such as cancer cell migration and invasion, underscoring the critical role of U1 homeostasis and suggesting it as a potential target for therapies. We describe a complex of U1 with cleavage and polyadenylation factors that silences PASs in introns and 3' UTR, which gives insights into U1 telescripting mechanism and transcription elongation regulation.

3'-UTRs and the Control of Protein Expression in Space and Time.

The noncoding elements of an mRNA influence multiple aspects of its fate. For example, 3'-UTRs serve as physical and sequence-based information hubs that direct the time, place, and level of translation of the protein encoded in cis, but often also have additional roles in trans. Understanding the information content of 3'-UTRs has been a challenge. Bioinformatic searches for motifs, such as those that encode the polyadenylation signal or microRNA seed regions, are simple enough, but rarely do these inferred positions in genomes correlate well with the actual sites chosen by the relevant nanomachines in living cells. This is almost certainly due to three-dimensional complexity of RNA, the physical states of which are recognized by RNA-binding proteins that serve to read and interpret the information content. Here, we follow the 3'-UTR-mediated posttranscriptional metabolism of mRNA in the germline of the nematode worm Caenorhabditis elegans. While many areas still require the clarification only detailed fundamental research can provide, this model system can serve as a basis of 3'-mediated regulatory control for elaboration in more complex metazoan systems.

A tale of two genes: divergent evolutionary fate of haptoglobin and hemopexin in hemoglobinless Antarctic icefishes.

The evolution of Antarctic notothenioid fishes in the isolated freezing Southern Ocean has led to remarkable trait gains and losses. One of the most extraordinary was the loss of the major oxygen carrier hemoglobin (Hb) in the icefishes (family Channichthyidae). Although the mechanisms of this loss and the resulting compensatory changes have been well studied, the impact of Hb loss on the network of genes that once supported its recycling and disposal has remained unexplored. Here, we report the functional fate and underlying molecular changes of two such key Hb-supporting proteins across the icefish family - haptoglobin (Hp) and hemopexin (Hx), crucial in removing cytotoxic free Hb and heme, respectively. Hp plays a critical role in binding free Hb for intracellular recycling and absent its primary client, icefish Hp transcription is now vanishingly little, and translation into a functional protein is nearly silenced. Hp genotype degeneration has manifested in separate lineages of the icefish phylogeny with three distinct nonsense mutations and a deletion frame shift, as well as mutated polyadenylation signal sequences. Thus, Hb loss appears to have diminished selective constraint on Hp maintenance, resulting in its stochastic, co-evolutionary drift towards extinction. Hx binds free heme for iron recycling in hepatocytes. In contrast to Hp, Hx genotype integrity is preserved in the icefishes and transcription occurs at levels comparable to those in the red-blooded notothenioids. The persistence of Hx likely owes to continued selective pressure for its function from mitochondrial and non-Hb cellular hemoproteins.

A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats

DNA sequences that read the same from 5′ to 3′ in either strand are called inverted repeat sequences or simply IRs. They are found throughout a wide variety of genomes, from prokaryotes to eukaryotes. Despite extensive research, their in vivo functions, if any, remain unclear. Using Saccharomyces cerevisiae, we performed genome-wide analyses for the distribution, occurrence frequency, sequence characteristics and relevance to chromatin structure, for the IRs that reportedly have a cruciform-forming potential. Here, we provide the first comprehensive map of these IRs in the S. cerevisiae genome. The statistically significant enrichment of the IRs was found in the close vicinity of the DNA positions corresponding to polyadenylation [poly(A)] sites and ~ 30 to ~ 60 bp downstream of start codon-coding sites (referred to as ‘start codons’). In the former, ApT- or TpA-rich IRs and A-tract- or T-tract-rich IRs are enriched, while in the latter, different IRs are enriched. Furthermore, we found a strong structural correlation between the former IRs and the poly(A) signal. In the chromatin formed on the gene end regions, the majority of the IRs causes low nucleosome occupancy. The IRs in the region ~ 30 to ~ 60 bp downstream of start codons are located in the + 1 nucleosomes. In contrast, fewer IRs are present in the adjacent region downstream of start codons. The current study suggests that the IRs play similar roles in Escherichia coli and S. cerevisiae to regulate or complete transcription at the RNA level.

Exons of Leukemia Suppressor Genes: Creative Assembly Required

Alternative splicing (AS) has many important roles in the pathogenesis of leukemia. Recent papers suggest that one of its key aspects is exclusion of 3'-terminal exons in favor of premature termination using intronic polyadenylation signals. This process generates leukemia suppressor isoforms with truncated C termini and acting in loss-of-function or dominant-negative manners.

A combinatorial code for mRNA 3'-UTR-mediated translational control in the mouse oocyte.

Meiotic maturation of mammalian oocytes depends on the temporally and spatially regulated cytoplasmic polyadenylation and translational activation of maternal mRNAs. Cytoplasmic polyadenylation is controlled by cis-elements in the 3′-UTRs of mRNAs including the polyadenylation signal (PAS), which is bound by the cleavage and polyadenylation specificity factor (CPSF) and the cytoplasmic polyadenylation element (CPE), which recruits CPE binding proteins. Using the 3′-UTRs of mouse Cpeb1, Btg4 and Cnot6l mRNAs, we deciphered the combinatorial code that controls developmental stage-specific translation during meiotic maturation: (i) translation of a maternal transcript at the germinal vesicle (GV) stage requires one or more PASs that locate far away from CPEs; (ii) PASs distal and proximal to the 3′-end of the transcripts are equally effective in mediating translation at the GV stage, as long as they are not close to the CPEs; (iii) Both translational repression at the GV stage and activation after germinal vesicle breakdown require at least one CPE adjacent to the PAS; (iv) The numbers and positions of CPEs in relation to PASs within the 3′-UTR of a given transcript determines its repression efficiency in GV oocytes. This study reveals a previously unrecognized non-canonical mechanism by which the proximal PASs mediate 3′-terminal polyadenylation and translation of maternal transcripts.

Repurposing of promoters and enhancers during mammalian evolution

Promoters and enhancers—key controllers of gene expression—have long been distinguished from each other based on their function. However, recent work suggested that common architectural and functional features might have facilitated the conversion of one type of element into the other during evolution. Here, based on cross-mammalian analyses of epigenome and transcriptome data, we provide support for this hypothesis by detecting 445 regulatory elements with signatures of activity turnover (termed P/E elements). Most events represent transformations of putative ancestral enhancers into promoters, leading to the emergence of species-specific transcribed loci or 5′ exons. Distinct GC sequence compositions and stabilizing 5′ splicing (U1) regulatory motif patterns may have predisposed P/E elements to regulatory repurposing, and changes in the U1 and polyadenylation signal densities and distributions likely drove the evolutionary activity switches. Our work suggests that regulatory repurposing facilitated regulatory innovation and the origination of new genes and exons during evolution.

Therapeutic Genome Editing for Myotonic Dystrophy Type 1Using CRISPR/Cas9.

Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3′ UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3′ UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3′ UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3′ UTR is a viable approach to develop therapeutic genome editing for DM1.

DeepGSR: an optimized deep-learning structure for the recognition of genomic signals and regions.

MotivationRecognition of different genomic signals and regions (GSRs) in DNA is crucial for understanding genome organization, gene regulation, and gene function, which in turn generate better genome and gene annotations. Although many methods have been developed to recognize GSRs, their pure computational identification remains challenging. Moreover, various GSRs usually require a specialized set of features for developing robust recognition models. Recently, deep-learning (DL) methods have been shown to generate more accurate prediction models than ‘shallow’ methods without the need to develop specialized features for the problems in question. Here, we explore the potential use of DL for the recognition of GSRs.ResultsWe developed DeepGSR, an optimized DL architecture for the prediction of different types of GSRs. The performance of the DeepGSR structure is evaluated on the recognition of polyadenylation signals (PAS) and translation initiation sites (TIS) of different organisms: human, mouse, bovine and fruit fly. The results show that DeepGSR outperformed the state-of-the-art methods, reducing the classification error rate of the PAS and TIS prediction in the human genome by up to 29% and 86%, respectively. Moreover, the cross-organisms and genome-wide analyses we performed, confirmed the robustness of DeepGSR and provided new insights into the conservation of examined GSRs across species.Availability and implementationDeepGSR is implemented in Python using Keras API; it is available as open-source software and can be obtained at https://doi.org/10.5281/zenodo.1117159.Supplementary information Supplementary data are available at Bioinformatics online.

Preliminary Findings of Structure and Expression of Opioid Receptor Genes in a Peregrine Falcon ( Falco peregrinus), a Snowy Owl ( Bubo scandiacus), and a Blue-fronted Amazon Parrot ( Amazona aestiva).

To further knowledge of the physiology of opioid receptors in birds, the structure and expression of the μ-, δ-, and κ-opioid receptor genes were studied in a peregrine falcon ( Falco peregrinus), a snowy owl ( Bubo scandiacus), and a blue-fronted Amazon parrot ( Amazona aestiva). Tissue samples were obtained from birds that had been euthanatized for poor release prognosis or medical reasons. Samples were taken from the brain (telencephalon, thalamus, pituitary gland, cerebellum, pons, medulla oblongata, mesencephalon), the spinal cord and dorsal root ganglions, and plantar foot skin. Messenger RNA was recovered, and reverse transcription polymerase chain reaction (RT-PCR) was performed to generate complementary DNA (cDNA) sequences. Gene structures were documented by directly comparing cDNA sequences with recently published genomic sequences for the peregrine falcon and the blue-fronted Amazon parrot or by comparisons with genomic sequences of related species for the snowy owl. Structurally, the avian μ-opioid receptor messenger RNA (mRNA) species were complex, displaying differential splicing, alternative stop codons, and multiple polyadenylation signals. In comparison, the structure of the avian κ-receptor mRNA was relatively simple. In contrast to what is seen in humans, the avian δ-receptor mRNA structure was found to be complex, demonstrating novel 3-prime coding and noncoding exons not identified in mammals. The role of the δ-opioid receptor merits further investigation in avian species.

Characterization of mRNA polyadenylation in the apicomplexa.

Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15–20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two—poly(A) polymerase and CPSF73—of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages–extracellular merozoites and intracellular schizonts–studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development.

Molecular characterisation of oestrogen receptor ERα and the effects of bisphenol A on its expression during sexual development in the Chinese giant salamander (Andrias davidianus).

The aim of this study was to characterise the molecular structure of the oestrogen receptor ERα and to evaluate the effect of bisphenol A (BPA) on ERα expression during sexual development of the Chinese giant salamander (Andrias davidianus). The ERα cDNA of A. davidianus includes an open reading frame of 1755bp (encoding 584 amino acids), a 219-bp 5' untranslated region (UTR) and a 611-bp 3'UTR. A polyadenylation signal was not found in the 3'UTR. Amino acid sequence analysis showed high homology between ERα of A. davidianus and that of other amphibians, such as Andrias japonicas (99.66% identity) and Rana rugose (81.06% identity). In 3-year-old A. davidianus, highest ERα expression was observed in the liver and gonads. During different developmental stages in A. davidianus (from 1 to 3 years of age), ERα expression in the testes increased gradually. ERα was localised in the epithelial cells of seminiferous lobules and in interstitial cells. ERα-positive cells were more abundant in the interstitial tissue during testicular development. ERα was located in the nucleus of oocytes during ovary development. We found that the sex of 6-month-old A. davidianus larvae could not be distinguished anatomically. The sex ratio did not change after larvae were treated with 10μM BPA for 1 month. However, BPA treatment reduced bodyweight and ERα expression in the gonads in male larvae.

Abstract 974: Alternative polyadenylation of androgen receptor variants in castration-resistant prostate cancer

Abstract The androgen receptor (AR), which is activated by androgens, functions in the normal prostate as a master transcriptional regulator. Accordingly, prostate cancer (PCa) presents as an androgen- and AR-dependent disease. Inhibition of AR transcriptional activity with androgen-deprivation therapy (ADT) is effective for patients with locally advanced and metastatic disease. All ADT suppresses AR via interference with its ligand binding domain (LBD)-dependent transcriptional activity. However, development of therapy resistance and transition to castration-resistant prostate cancer (CRPC) represents a major clinical challenge. CRPC circumvents ADT by expressing constitutively active AR variants (AR-Vs) that lack the LBD. Therapies targeting constitutively active AR variants are not currently available. To address this problem, we investigated the mechanisms underlying AR-V expression in CRPC. One particular AR-V, called AR-V7, consists of contiguously spliced exons 1, 2, 3, and a cryptic exon, CE3, and has been shown to be associated with clinical resistance to second-generation AR inhibitors abiraterone and enzalutamide. We noted that CE3 contains a consensus hexameric AAUAAA polyadenylation signal (PAS). We hypothesized that intronic polyadenylation promotes AR-V7 expression in CRPC. To test this, we developed a morpholino that sterically blocked the CE3 PAS to prevent splicing of AR-V7. We have demonstrated that the PAS in CE3 controls expression of AR-V7, as well as a broad array of AR variants arising by splicing of adjacent cryptic exons that use the shared CE3 PAS. Furthermore, our data demonstrate that the cleavage and polyadenylation complex (CPSF complex) is enriched in primary PCa tumors relative to normal tissues. We have also demonstrated that CPSF1, a member of the CPSF complex, controls expression of AR-Vs relative to full length AR and plays a global role in the determination of mRNA isoform length in prostate cancer cells. Collectively, these findings reveal alterations in the cellular machinery governing mRNA cleavage and polyadenylation in prostate cancer, and identify specific opportunities for therapeutic intervention. Citation Format: Jamie L. Van Etten, Michael Nyquist, Yingming Li, Rebecca LaRue, Yeung Ho, Rendong Yang, Daniel F. Voytas, Christine Henzler, Scott M. Dehm. Alternative polyadenylation of androgen receptor variants in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 974.

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling.

The Wnt signaling pathway can be grouped into two classes, the β-catenin-dependent and β-catenin-independent pathways. Wnt5a signaling through a β-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and safeguard transcriptome integrity1. Pol II accumulation downstream of the cleavage and polyadenylation signal (CPS) can facilitate recruitment of factors involved in mRNA 3’-end formation and termination2, but how this sequence is initiated remains unclear. In a chemical-genetic screen, we identified human protein phosphatase 1 (PP1) isoforms as substrates of positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex3. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis24. Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain (CTD) can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after Cdk9 inhibition in vivo. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, concomitant with accumulation of Pol II phosphorylated at CTD repeat residue Ser25. A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq)6, and is genetically suppressed by ablation of Cdk9 target sites in Spt5. These results suggest that the transition from elongation to termination by Pol II coincides with Dis2-dependent reversal of Cdk9 signaling—a switch analogous to a Cdk1-PP1 circuit that controls mitotic progression4.

Alternative Polyadenylation of Mammalian Transcripts Is Generally Deleterious, Not Adaptive.

Alternative polyadenylation (APA) produces from thesame gene multiple mature RNAs with varying 3' ends. Although APA is commonly believed to generate beneficial functional diversity and be adaptive, we hypothesize that most genes have one optimal polyadenylation site and that APA is caused largely by deleterious polyadenylation errors. The error hypothesis, but not the adaptive hypothesis, predicts that, as the expression level of a gene increases, its polyadenylation diversity declines, relative use of the major (presumably optimal) polyadenylation site increases, and that of each minor (presumably nonoptimal) site decreases. It further predicts that the number of polyadenylation signals per gene is smaller than the random expectation and that polyadenylation signals for major but not minor sites are under purifying selection. All of these predictions are confirmed in mammals, suggesting that numerous defective RNAs are produced in normal cells, many phenotypic variations at the molecular level are nonadaptive, and cellular life is noisier than is appreciated.

Adenosine causes read-through into the late region of the HPV16 genome in a guanosine-dependent manner

Adenosine plays an important role in cell death and differentiation as well as in tumorigenesis and the intra- and extra-cellular levels range from nanomolar to millimolar levels under various physiological or pathophysiological conditions. Here we report that adenosine can activate HPV16 late gene expression in a dose- and time-dependent manner, but only in the presence of guanosine. This activation occurred within hours after addition of the nucleosides and was primarily dependent on the ENT1 nucleoside transporter protein. Induction of HPV16 late gene expression was mainly the result of increased read-through at the early HPV16 polyadenylation signal into the late region of the HPV16 genome, thereby producing HPV16 late L2 mRNAs. The effect of guanosine and adenosine on HPV16 late gene expression was mediated by the increased binding to HPV16 mRNAs and nuclear export of the cellular HuR protein. Our results demonstrate that nucleosides can affect HPV16 gene expression.

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

BackgroundThe Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA.MethodsIn order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed.ResultsWe show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear “scrambled” whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter.ConclusionAltogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Peculiar genes for thermostable bifunctional catalase-peroxidases in Chaetomium thermophilum and their molecular evolution

Catalase-peroxidases represent one important subfamily of ancestral antioxidant enzymes originally evolved in bacteria for the protection against various forms of oxidative stress. KatG genes coding for these bifunctional catalase-peroxidases were during their peculiar evolution transferred from Bacteroidetes to the fungal phylum Ascomycota via a horizontal gene transfer event. Here we analyse a newly discovered fungal katG gene without introns coding for a thermostable catalase-peroxidase from Chaetomium thermophilum var. dissitum and compare it with closely related thermophilic and mesophilic katGs and their translation products. We show that CthediskatG gene resembling its bacterial counterparts has a typical eukaryotic transcription start site and also contains a conserved eukaryotic polyadenylation signal behind its 3′ terminus. Moreover, we have detected polyA tails in corresponding transcripts of katG from two different mRNA libraries of C. thermophilum var. disstum. Although otherwise highly conserved, only in katG genes of two C. thermophilum variants a unique 60 bp long deletion leading in the translated product with high probability to a modified loop and thus access to the prosthetic heme group was observed. We also present an updated molecular phylogeny revealing the evolutionary position of fungal thermostable catalase-peroxidases within a robust phylogenetic tree of the whole KatG subfamily.