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Interferon (IFN) induced activities are critical, early determinants of immune responses and infection outcomes. A key facet of IFN responses is the upregulation of hundreds of mRNAs termed interferon-stimulated genes (ISGs) that activate intrinsic and cell-mediated defenses. While primary interferon signaling is well-delineated, other layers of regulation are less explored but implied by aberrant ISG expression signatures in many diseases in the absence of infection. Consistently, our examination of tonic ISG levels across uninfected human tissues and individuals revealed three ISG subclasses. As tissue identity and many comorbidities with increased virus susceptibility are characterized by differences in metabolism, we characterized ISG responses in cells grown in media known to favor either aerobic glycolysis (glucose) or oxidative phosphorylation (galactose supplementation). While these conditions over time had a varying impact on the expression of ISG RNAs, the differences were typically greater between treatments than between glucose/galactose. Interestingly, extended interferon-priming led to divergent expression of two ISG proteins: upregulation of IRF1 in IFN-γ/glucose and increased IFITM3 in galactose by IFN-α and IFN-γ. In agreement with a hardwired response, glucose/galactose regulation of interferon-γ induced IRF1 is conserved in unrelated mouse and cat cell types. In galactose conditions, proteasome inhibition restored interferon-γ induced IRF1 levels to that of glucose/interferon-γ. Glucose/interferon-γ decreased replication of the model poxvirus vaccinia at low MOI and high MOIs. Vaccinia replication was restored by IRF1 KO. In contrast, but consistent with differential regulation of IRF1 protein by glucose/galactose, WT and IRF1 KO cells in galactose media supported similar levels of vaccinia replication regardless of IFN-γ priming. Also associated with glucose/galactose is a seemingly second block at a very late stage in viral replication which results in reductions in herpes- and poxvirus titers but not viral protein expression. Collectively, these data illustrate a novel layer of regulation for the key ISG protein, IRF1, mediated by glucose/galactose and imply unappreciated subprograms embedded in the interferon response. In principle, such cellular circuitry could rapidly adapt immune responses by sensing changing metabolite levels consumed during viral replication and cell proliferation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Proper recognition of viral pathogens is an essential part of the innate immune response. A common viral replicative intermediate and chemical signal that cells use to identify pathogens is the presence of a triphosphorylated 5' end (5'ppp) RNA, which activates the cytosolic RNA sensor RIG-I and initiates downstream antiviral signaling. While 5'pppRNA generated by viral RNA-dependent RNA polymerases (RdRps) can be a potent activator of the immune response, endogenous RNA polymerase III (RNAPIII) transcripts can retain the 5'pppRNA generated during transcription and induce a RIG-I-mediated immune response. We have previously shown that host RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) can act on both host and viral RNAs, altering their levels and reducing their ability to induce RIG-I activation. Our previous work explored how artificially altered DUSP11 can impact immune activation, prompting further exploration into natural contexts of altered DUSP11. Here, we have identified viral DUSP11 homologs (vDUSP11s) present in some avipoxviruses. Consistent with the known functions of endogenous DUSP11, we have shown that expression of vDUSP11s: 1) reduces levels of endogenous RNAPIII transcripts, 2) reduces a cell's sensitivity to 5'pppRNA-mediated immune activation, and 3) restores virus infection defects seen in the absence of DUSP11. Our results identify a virus-relevant context where DUSP11 activity has been co-opted to alter RNA metabolism and influence the outcome of infection.

Inflammasomes are multiprotein complexes that form in response to ligands originating from pathogens as well as alterations of normal cell physiology caused by infection or tissue damage. These structures engage a robust inflammatory immune response that eradicates environmental microbes before they cause disease, and slow the growth of bona fide pathogens. Despite their undeniable utility in immunity, inflammasomes are radically reduced in birds. Perhaps most surprising is that, within all birds, NLRP3 is retained, while its signaling adapter ASC is lost, suggesting that NLRP3 signals via a novel unknown adapter. Crocodilian reptiles and turtles, which share a more recent common ancestor with birds, retain many of the lost inflammasome components, indicating that the deletion of inflammasomes occurred after birds diverged from crocodiles. Some bird lineages have even more extensive inflammasome loss, with songbirds continuing to pare down their inflammasomes until only NLRP3 and CARD8 remain. Remarkably, songbirds have lost caspase-1 but retain the downstream targets of caspase-1: IL-1β, IL-18, and the YVAD-linker encoding gasdermin A. This suggests that inflammasomes can signal through alternative proteases to activate cytokine maturation and pyroptosis in songbirds. These observations may reveal new contexts of activation that may be relevant to mammalian inflammasomes and may suggest new avenues of research to uncover the enigmatic nature of the poorly understood NLRP3 inflammasome., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The phylum Nucleocytoviricota includes the largest and most complex viruses known. These "giant viruses" have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host-referred to here as viral homologs or "virologs"-as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery "imitates" immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis 1-3 . Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

Mitochondria are dynamic organelles vital for energy production with now appreciated roles in immune defense. During microbial infection, mitochondria serve as signaling hubs to induce immune responses to counteract invading pathogens like viruses. Mitochondrial functions are central to a variety of antiviral responses including apoptosis and type I interferon signaling (IFN-I). While apoptosis and IFN-I mediated by m itochondrial a ntiviral s ignaling (MAVS) are well-established defenses, new dimensions of mitochondrial biology are emerging as battlefronts during viral infection. Increasingly, it has become apparent that mitochondria serve as reservoirs for distinct cues that trigger immune responses and that alterations in mitochondrial morphology may also tip infection outcomes. Furthermore, new data are foreshadowing pivotal roles for classic, homeostatic facets of this organelle as host-virus interfaces, namely, the t ri c arboxylic a cid (TCA) cycle and e lectron t ransport c hain (ETC) complexes like respiratory supercomplexes. Underscoring the importance of "housekeeping" mitochondrial activities in viral infection is the growing list of viral-encoded inhibitors including mimics derived from cellular genes that antagonize these functions. For example, virologs for ETC factors and several enzymes from the TCA cycle have been recently identified in DNA virus genomes and serve to pinpoint new vulnerabilities during infection. Here, we highlight recent advances for known antiviral functions associated with mitochondria as well as where the next battlegrounds may be based on viral effectors. Collectively, new methodology and mechanistic insights over the coming years will strengthen our understanding of how an ancient molecular truce continues to defend cells against viruses.

Pathogen infection triggers host innate defenses which may result in the activation of regulated cell death (RCD) pathways such as apoptosis. Given a vital role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptosis. Yet, it is unclear whether components of this emerging RCD pathway display signatures associated with pathogen conflict that are rare in combination but common to key host defense factors, namely, rapid evolution, viral homolog (virolog), and cytokine induction. We leveraged evolutionary sequence analysis that examines rates of amino acid replacement, which revealed: 1) strong and recurrent signatures of positive selection for primate and bat RIPK3 and MLKL, and 2) elevated rates of amino acid substitution on multiple RIPK3/MLKL surfaces suggestive of past antagonism with multiple, distinct pathogen-encoded inhibitors. Furthermore, our phylogenomics analysis across poxvirus genomes illuminated volatile patterns of evolution for a recently described MLKL viral homolog. Specifically, poxviral MLKLs have undergone numerous gene replacements mediated by duplication and deletion events. In addition, MLKL protein expression is stimulated by interferons in human and mouse cells. Thus, MLKL displays all three hallmarks of pivotal immune factors of which only a handful of factors like OAS1 exhibit. These data support the hypothesis that over evolutionary time MLKL functions-which may include execution of necroptosis-have served as a major determinant of infection outcomes despite gene loss in some host genomes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Host-pathogen conflicts leave genetic signatures in genes that are critical for host defense functions. Using these "molecular scars" as a guide to discover gene functions, we discovered a vertebrate-specific MItochondrial STress Response (MISTR) circuit. MISTR proteins are associated with electron transport chain (ETC) factors and activated by stress signals such as interferon gamma (IFNγ) and hypoxia. Upon stress, ultraconserved microRNAs (miRNAs) down-regulate MISTR1(NDUFA4) followed by replacement with paralogs MItochondrial STress Response AntiViral (MISTRAV) and/or MItochondrial STress Response Hypoxia (MISTRH). While cells lacking MISTR1(NDUFA4) are more sensitive to chemical and viral apoptotic triggers, cells lacking MISTRAV or expressing the squirrelpox virus-encoded vMISTRAV exhibit resistance to the same insults. Rapid evolution signatures across primate genomes for MISTR1(NDUFA4) and MISTRAV indicate recent and ongoing conflicts with pathogens. MISTR homologs are also found in plants, yeasts, a fish virus, and an algal virus indicating ancient origins and suggesting diverse means of altering mitochondrial function under stress. The discovery of MISTR circuitry highlights the use of evolution-guided studies to reveal fundamental biological processes., Competing Interests: The authors have declared that no competing interests exist.

Moebius syndrome (MBS) is a congenital disorder caused by paralysis of the facial and abducens nerves. Although a number of candidate genes have been suspected, so far only mutations in PLXND1 and REV3L are confirmed to cause MBS. Here, we fine mapped the breakpoints of a complex chromosomal rearrangement (CCR) 46,XY,t(7;8;11;13) in a patient with MBS, which revealed 41 clustered breakpoints with typical hallmarks of chromothripsis. Among 12 truncated protein-coding genes, SEMA3A is known to bind to the MBS-associated PLXND1. Intriguingly, the CCR also truncated PIK3CG, which in silico interacts with REVL3 encoded by the other known MBS-gene REV3L, and with the SEMA3A/PLXND1 complex via FLT1. Additional studies of other complex rearrangements may reveal whether the multiple breakpoints in germline chromothripsis may predispose to complex multigenic disorders., (© 2019 Wiley Periodicals, Inc.)

Chromothripsis is a mutational event driven by tens to hundreds of double-stranded DNA breaks which occur in a single event between a limited number of chromosomes. Following chromosomal shattering, DNA fragments are stitched together in a seemingly random manner resulting in complex genomic rearrangements including sequence shuffling, deletions, and inversions of varying size. This genomic catastrophe has been observed in cancer genomes and the genomes of patients harboring developmental and congenital defects. The mechanisms catalyzing DNA breakage and coordinating the "random" assembly of genomic fragments are actively being investigated. Recently, retrotransposons-a type of "jumping gene"-have been implicated as one means to generate double-stranded DNA breaks during chromothripsis and as sequences which can contribute to the final configuration of the derived chromosomes. In this methods chapter, I discuss how to apply available bioinformatic tools and the hallmarks of retrotransposon mobilization to breakpoint junctions to assess the role for active and inactive retrotransposon sequences in chromothriptic events.

Background: Recent reports indicate that retrotransposons - a type of mobile DNA - can contribute to neuronal genetic diversity in mammals. Retrotransposons are genetic elements that mobilize via an RNA intermediate by a "copy-and-paste" mechanism termed retrotransposition. Long Interspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in humans and its activity is responsible for ~ 30% of genomic mass. Historically, L1 retrotransposition was thought to be restricted to the germline; however, new data indicate L1 s are active in somatic tissue with certain regions of the brain being highly permissive. The functional implications of L1 insertional activity in the brain and how host cells regulate it are incomplete. While deep sequencing and qPCR analysis have shown that L1 copy number is much higher in certain parts of the human brain, direct in vivo studies regarding detection of L1-encoded proteins is lacking due to ineffective reagents., Results: Using a polyclonal antibody we generated against the RNA-binding (RRM) domain of L1 ORF1p, we observe widespread ORF1p expression in post-mortem human brain samples including the hippocampus which has known elevated rates of retrotransposition. In addition, we find that two brains from different individuals of different ages display very different expression of ORF1p, especially in the frontal cortex., Conclusions: We hypothesize that discordance of ORF1p expression in parts of the brain reported to display elevated levels of retrotransposition may suggest the existence of factors mediating post-translational regulation of L1 activity in the human brain. Furthermore, this antibody reagent will be useful as a complementary means to confirm findings related to retrotransposon biology and activity in the brain and other tissues in vivo.

Over evolutionary time, the dynamic nature of a genome is driven, in part, by the activity of transposable elements (TE) such as retrotransposons. On a shorter time scale it has been established that new TE insertions can result in single-gene disease in an individual. In humans, the non-LTR retrotransposon Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous TE. In addition to mobilizing its own RNA to new genomic locations via a "copy-and-paste" mechanism, LINE-1 is able to retrotranspose other RNAs including Alu, SVA, and occasionally cellular RNAs. To date in humans, 124 LINE-1-mediated insertions which result in genetic diseases have been reported. Disease causing LINE-1 insertions have provided a wealth of insight and the foundation for valuable tools to study these genomic parasites. In this review, we provide an overview of LINE-1 biology followed by highlights from new reports of LINE-1-mediated genetic disease in humans.

Chromothripsis (CTH) is a phenomenon where multiple localized double-stranded DNA breaks result in complex genomic rearrangements. Although the DNA-repair mechanisms involved in CTH have been described, the mechanisms driving the localized "shattering" process remain unclear. High-throughput sequence analysis of a familial germline CTH revealed an inserted SVAE retrotransposon associated with a 110-kb deletion displaying hallmarks of L1-mediated retrotransposition. Our analysis suggests that the SVAE insertion did not occur prior to or after, but concurrent with the CTH event. We also observed L1-endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110-kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity facilitating DNA cleavage by catalytically active L1-endonuclease. Our data provide the first evidence that active and inactive human retrotransposons can serve as endogenous mutagens driving CTH in the germline., (© 2016 WILEY PERIODICALS, INC.)

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular 'arms races.' Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2'-5'-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.

Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.

Long INterspersed Elements (LINE-1s, L1s) are responsible for over one million retrotransposon insertions and 8000 processed pseudogenes (PPs) in the human genome. An active L1 encodes two proteins (ORF1p and ORF2p) that bind with L1 RNA and form L1-ribonucleoprotein particles (RNPs). Although it is believed that the RNA-binding property of ORF1p is critical to recruit other mobile RNAs to the RNP, the identity of recruited RNAs is largely unknown. Here, we used crosslinking and immunoprecipitation followed by deep sequencing to identify RNA components of L1-RNPs. Our results show that in addition to retrotransposed RNAs [L1, Alu and SINE-VNTR-Alu (SVA)], L1-RNPs are enriched with cellular mRNAs, which have PPs in the human genome. Using purified L1-RNPs, we show that PP-source RNAs preferentially serve as ORF2p templates in a reverse transcriptase assay. In addition, we find that exogenous ORF2p binds endogenous ORF1p, allowing reverse transcription of the same PP-source RNAs. These data demonstrate that interaction of a cellular RNA with the L1-RNP is an inside track to PP formation.

RNA-based duplication mediated by reverse transcriptase (RT), a process termed retrotransposition, is ongoing in humans and is a source of significant inter- and perhaps intraindividual genomic variation. The long interspersed element 1 (LINE-1 or L1) ORF2 protein is the genomic source for RT activity required for mobilization of its own RNA in cis and other RNAs, such as SINE/variable-number tandem-repeat (VNTR)/Alu (SVA) elements, in trans. SVA elements are ~2-kb hominid-specific noncoding RNAs that have resulted in single-gene disease in humans through insertional mutagenesis or aberrant mRNA splicing. Here, using an SVA retrotransposition cell culture assay in U2OS cells, we investigated SVA domains important in L1-mediated SVA retrotransposition. Partial- and whole-domain deletions revealed that removal of either the Alu-like or SINE-R domain in the context of a full-length SVA has little to no effect, whereas removal of the CT hexamer or the VNTR domain can result in a 75% decrease in activity. Additional experiments demonstrate that the Alu-like fragment alone can retrotranspose at low levels while the addition of the CT hexamer can enhance activity as much as 2-fold compared to that of the full-length SVA. These results suggest that no SVA domain is essential for retrotransposition in U2OS cells and that the 5' end of SVA (hexamer and Alu-like domain) is sufficient for retrotransposition.

Mobile DNAs, also known as transposons or 'jumping genes', are widespread in nature and comprise an estimated 45% of the human genome. Transposons are divided into two general classes based on their transposition intermediate (DNA or RNA). Only one subclass, the non-LTR retrotransposons, which includes the Long INterspersed Element-1 (LINE-1 or L1), is currently active in humans as indicated by 96 disease-causing insertions. The autonomous LINE-1 is capable of retrotransposing not only a copy of its own RNA in cis but also other RNAs (Alu, SINE-VNTR-Alu (SVA), U6) in trans to new genomic locations through an element encoded reverse transcriptase. L1 can also retrotranspose cellular mRNAs, resulting in processed pseudogene formation. Here, we highlight recent reports that update our understanding of human L1 retrotransposition and their role in disease. Finally we discuss studies that provide insights into the past and current activity of these retrotransposons, and shed light on not just when, but where, retrotransposition occurs and its part in genetic variation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Long INterspersed Element-1 (LINE-1) retrotransposons comprise 17% of the human genome, and move by a potentially mutagenic "copy and paste" mechanism via an RNA intermediate. Recently, the retrotransposition-mediated insertion of a new transcript was described as a novel cause of genetic disease, Duchenne muscular dystrophy, in a Japanese male. The inserted sequence was presumed to derive from a single-copy, noncoding RNA transcribed from chromosome 11q22.3 that retrotransposed into the dystrophin gene. Here, we demonstrate that a nonreference full-length LINE-1 is situated in the proband and maternal genome at chromosome 11q22.3, directly upstream of the sequence, whose copy was inserted into the dystrophin gene. This LINE-1 is highly active in a cell culture assay. LINE-1 insertions are often associated with 3' transduction of adjacent genomic sequences. Thus, the likely explanation for the mutagenic insertion is a LINE-1-mediated 3' transduction with severe 5' truncation. This is the first example of LINE-1-induced human disease caused by an "orphan" 3' transduction., (© 2011 Wiley Periodicals, Inc.)

Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5' and 3' transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5' end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed.

SINE-VNTR-Alus (SVA) are non-autonomous hominid specific retrotransposons that are associated with disease in humans. SVAs are evolutionarily young and presumably mobilized by the LINE-1 reverse transcriptase in trans. SVAs are currently active and may impact the host through a variety of mechanisms including insertional mutagenesis, exon shuffling, alternative splicing, and the generation of differentially methylated regions (DMR). Here we review SVA biology, including SVA insertions associated with known diseases. Further, we discuss a model describing the initial formation of SVA and the mechanisms by which SVA may impact the host., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Although most human retrotransposons are inactive, both inactive and active retrotransposons drive genome evolution and may influence transcription through various mechanisms. In humans, three retrotransposon families are still active, but one of these, SVA, remains mysterious. Here we report the identification of a new subfamily of SVA, which apparently formed after an alternative splicing event where the first exon of the MAST2 gene spliced into an intronic SVA and subsequently retrotransposed. Additional examples of SVA retrotransposing upstream exons due to splicing into SVA were also identified in other primate genomes. After molecular and computational experiments, we found a number of functional 3' splice sites within many different transcribed SVAs across the human and chimpanzee genomes. Using a minigene splicing construct containing an SVA, we observed splicing in cell culture, along with SVA exonization events that introduced premature termination codons (PTCs). These data imply that an SVA residing within an intron in the same orientation as the gene may alter normal gene transcription either by gene-trapping or by introducing PTCs through exonization, possibly creating differences within and across species.

Variation in gene expression is a fundamental aspect of human phenotypic variation. Several recent studies have analyzed gene expression levels in populations of different continental ancestry and reported population differences at a large number of genes. However, these differences could largely be due to non-genetic (e.g., environmental) effects. Here, we analyze gene expression levels in African American cell lines, which differ from previously analyzed cell lines in that individuals from this population inherit variable proportions of two continental ancestries. We first relate gene expression levels in individual African Americans to their genome-wide proportion of European ancestry. The results provide strong evidence of a genetic contribution to expression differences between European and African populations, validating previous findings. Second, we infer local ancestry (0, 1, or 2 European chromosomes) at each location in the genome and investigate the effects of ancestry proximal to the expressed gene (cis) versus ancestry elsewhere in the genome (trans). Both effects are highly significant, and we estimate that 12+/-3% of all heritable variation in human gene expression is due to cis variants., Competing Interests: The authors have declared that no competing interests exist.

Fundulus notatus and Fundulus olivaceus are two closely related topminnow species that exhibit similar ecological niches and broad, largely overlapping, North American ranges extending throughout much of the Mississippi River drainage as well as the coastal drainages of the Gulf of Mexico. Previous studies have suggested that these two species are reproductively compatible despite cytogenetic differences and will hybridize when syntopic. We used nuclear and mtDNA loci to assess levels of hybridization and test for introgression in syntopic populations of these two species in four drainages in southern Illinois. Although hybridization was detected in all syntopic populations, an assessment of the proportion of hybrid individuals indicated a deficiency of hybrids relative to expectations under random mating. We determined that, although mtDNA introgression was prevalent and extended beyond the zones of contact, evidence of nuclear introgression was limited to the zone of sympatry.