Chimeric retrogenes suggest a role for the nucleolus in LINE amplification

AbstractChimeric retrogenes, found in mammalian and fungal genomes, are bipartite elements composed of DNA copies of cellular transcripts either directly fused to each other or fused to the 3′ part of a LINE retrotransposon. These cellular transcripts correspond to messenger RNAs, ribosomal RNAs, small nuclear RNAs and 7SL RNA. The chimeras are likely formed by RNA template switches during reverse transcription of LINE elements by their retrotranspositional machinery. The 5′ part of chimeras are copies of nucleolar RNAs, suggesting that the nucleolus plays a significant role in LINE retrotransposition. RNAs from the nucleolus might have protective function against retroelement invasion or, alternatively, the nucleolus may be required for retrotranspositional complex assembly and maturation. These hypotheses will be discussed in this review

Functional human endogenous retroviral LTR transcription start sites are located between the R and U5 regions

AbstractHuman endogenous retroviruses (HERVs) occupy about 5% of human DNA and are thought to be remnants of ancient retroviral infections of human ancestors' germ cells. HERVs can modify expression of host cell genes through their cis-regulatory elements concentrated in their long terminal repeats (LTRs). Although numerous HERV-related RNAs were identified in the human transcriptome, for most of them, it remains unclear whether they are LTR-promoted or read-through products initiated from neighboring genomic promoters. Here, we describe mapping of transcriptional start sites within solitary and proviral LTRs of the HERV-K (HML-2) human-specific subfamily of endogenous retroviruses. Surprisingly, the transcription was initiated predominantly from the very 3′ termini of the LTR R regions. The data presented here may shed light on adaptive coevolution of human endogenous retroviruses with their host cells

Tripartite chimeric pseudogene from the genome of rice blast fungus Magnaporthe grisea suggests double template jumps during long interspersed nuclear element (LINE) reverse transcription

<p>Abstract</p> <p>Background</p> <p>A systematic survey of loci carrying retrotransposons in the genome of the rice blast fungus <it>Magnaporthe grisea </it>allowed the identification of novel non-canonical retropseudogenes. These elements are chimeric retrogenes composed of DNA copies from different cellular transcripts directly fused to each other. Their components are copies of a non protein-coding highly expressed RNA of unknown function termed WEIRD and of two fungal retrotransposons: MGL and Mg-SINE. Many of these chimeras are transcribed in various <it>M. grisea </it>tissues and during plant infection. Chimeric retroelements with a similar structure were recently found in three mammalian genomes. All these chimeras are likely formed by RNA template switches during the reverse transcription of diverse LINE elements.</p> <p>Results</p> <p>We have shown that in <it>M. grisea </it>template switching occurs at specific sites within the initial template RNA which contains a characteristic consensus sequence. We also provide evidence that both single and double template switches may occur during LINE retrotransposition, resulting in the fusion of three different transcript copies. In addition to the 33 bipartite elements, one tripartite chimera corresponding to the fusion of three retrotranscripts (WEIRD, Mg-SINE, MGL-LINE) was identified in the <it>M. grisea </it>genome. Unlike the previously reported two human tripartite elements, this fungal retroelement is flanked by identical 14 bp-long direct repeats. The presence of these short terminal direct repeats demonstrates that the LINE enzymatic machinery was involved in the formation of this chimera and its integration in the <it>M. grisea </it>genome.</p> <p>Conclusion</p> <p>A survey of mammalian genomic databases also revealed two novel tripartite chimeric retroelements, suggesting that double template switches occur during reverse transcription of LINE retrotransposons in different eukaryotic organisms.</p

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death

Background: Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies.Results: Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55 degrees C and 85 degrees C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 degrees C, the only striking morphological difference between cell deaths induced by 40-55 degrees C or 85 degrees C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents.Conclusions: We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways

Novel strong tissue specific promoter for gene expression in human germ cells

<p>Abstract</p> <p>Background</p> <p>Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene <it>NDUFV1 </it>containing an endogenous retroviral sequence.</p> <p>Results</p> <p>Among seven established human cell lines and five primary cultures, this modified <it>NDUFV1 </it>upstream sequence (mNUS) was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102), where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter). To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD) suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1), whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293). In further experiments, we tested mNUS-driven expression of a hyperactive <it>Sleeping Beauty </it>transposase (SB100X). The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter.</p> <p>Conclusions</p> <p>We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream <it>NDUFV1 </it>transcriptional start site plays a crucial role in the activity of this gene promoter <it>in vitro </it>in the majority of tested cell types (10/12), and an important role - in the rest two cell lines.</p

Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis) or “negative” (silencing) mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1). Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation

At Least 50% of Human-Specific HERV-K (HML-2) Long Terminal Repeats Serve In Vivo as Active Promoters for Host Nonrepetitive DNA Transcription

We report the first genome-wide comparison of in vivo promoter activities of a group of human-specific endogenous retroviruses in healthy and cancerous germ line tissues. To this end, we employed a recently developed technique termed genomic repeat expression monitoring. We found that at least 50% of human-specific long terminal repeats (LTRs) possessed promoter activity, and many of them were up- or downregulated in a seminoma. Individual LTRs were expressed at markedly different levels, ranging from ∼0.001 to ∼3% of the housekeeping beta-actin gene transcript level. We demonstrated that the main factors affecting the LTR promoter activity were the LTR type (5′-proviral, 3′ proviral, or solitary) and position with regard to genes. The averaged promoter strengths of solitary and 3′-proviral LTRs were almost identical in both tissues, whereas 5′-proviral LTRs displayed two- to fivefold higher promoter activities. The relative content of promoter-active LTRs in gene-rich regions was significantly higher than that in gene-poor loci. This content was maximal in those regions where LTRs “overlapped” readthrough transcripts. Although many promoter-active LTRs were mapped near known genes, no clear-cut correlation was observed between transcriptional activities of genes and neighboring LTRs. Our data also suggest a selective suppression of transcription for LTRs located in gene introns

Improving specificity of DNA hybridization-based methods

Methods based on DNA reassociation in solution with the subsequent PCR amplification of certain hybrid molecules, such as coincidence cloning and subtractive hybridization, all suffer from a common imperfection: cross-hybridization between various types of paralogous repetitive DNA fragments. Although the situation can be slightly improved by the addition of repeat-specific competitor DNA into the hybridization mixture, the cross-hybridization outcome is a significant number of background chimeric clones in resulting DNA libraries. In order to overcome this challenge, we developed a technique called mispaired DNA rejection (MDR), which utilizes a treatment of resulting reassociated DNA with mismatch-specific nucleases. We examined the MDR efficiency using cross-hybridization of complex, whole genomic mixtures derived from human and chimpanzee genomes, digested with frequent-cutter restriction enzyme. We show here that both single-stranded DNA-specific and mismatched double-stranded DNA-specific nucleases can be used for MDR separately or in combination, reducing the background level from 60 to 4% or lower. The technique presented here is of universal usefulness and can be applied to both cDNA and genomic DNA subtractions of very complex DNA mixtures. MDR is also useful for the genome-wide recovery of highly conserved DNA sequences, as we demonstrate by comparing human and pygmy marmoset genomes