The sound of silence: mechanisms and implications of HUSH complex function.

The vertebrate genome is under constant threat of invasion by genetic parasites. Whether the host can immediately recognize and respond to invading elements has been unclear. The discovery of the human silencing hub (HUSH) complex, and the finding that it provides immediate protection from genome invasion by silencing products of reverse transcription, have important implications for mammalian genome evolution. In this review, we summarize recent insights into HUSH function and describe how cellular introns provide a novel means of self-nonself discrimination, allowing HUSH to recognize and transcriptionally repress a broad range of intronless genetic elements. We discuss how HUSH contributes to genome evolution, and highlight studies reporting the critical role of HUSH in development and implicating HUSH in the control of immune signaling and cancer progression.

When cleaning facilitates cluttering - genome editing in ciliates.

Many organisms remove DNA from their genomes during development. This has foremost been characterized as a means of defending genomes against mobile elements. However, genome editing actually hides such elements from purifying selection, with the survivors evolving approximately neutrally, 'cluttering' the germline genome, enabling it to enlarge over time.

Throwing away DNA: programmed downsizing in somatic nuclei.

Programmed elimination of DNA during development yields somatic cell nuclei with dramatically different DNA sequence and content relative to germline nuclei, profoundly influencing genome architecture and stability. Whole-genome sequencing has significantly expanded the list of taxa known to exhibit this trait and has revealed the identity of excised genes and transposable elements (TEs) in certain taxa. Here, we compare the diverse mechanisms employed by ciliates, nematodes, copepods, and lamprey to downsize their genomes during development and propose tests of hypotheses about the evolution and/or maintenance of this trait. We explore possible functional roles that programmed DNA elimination (PDE) could play in genomic defense (especially against TEs), regulation of development, sex determination, co-option, and modulating nucleotypic effects, which together argue for a place in the mainstream investigation of genome evolution.

The Role of DNA Methylation in Genome Defense in Cnidaria and Other Invertebrates.

Considerable attention has recently been focused on the potential involvement of DNA methylation in regulating gene expression in cnidarians. Much of this work has been centered on corals, in the context of changes in methylation perhaps facilitating adaptation to higher seawater temperatures and other stressful conditions. Although first proposed more than 30 years ago, the possibility that DNA methylation systems function in protecting animal genomes against the harmful effects of transposon activity has largely been ignored since that time. Here, we show that transposons are specifically targeted by the DNA methylation system in cnidarians, and that the youngest transposons (i.e., those most likely to be active) are most highly methylated. Transposons in longer and highly active genes were preferentially methylated and, as transposons aged, methylation levels declined, reducing the potentially harmful side effects of CpG methylation. In Cnidaria and a range of other invertebrates, correlation between the overall extent of methylation and transposon content was strongly supported. Present transposon burden is the dominant factor in determining overall level of genomic methylation in a range of animals that diverged in or before the early Cambrian, suggesting that genome defense represents the ancestral role of CpG methylation.

The RNAi Universe in Fungi: A Varied Landscape of Small RNAs and Biological Functions.

RNA interference (RNAi) is a conserved eukaryotic mechanism that uses small RNA molecules to suppress gene expression through sequence-specific messenger RNA degradation, translational repression, or transcriptional inhibition. In filamentous fungi, the protective function of RNAi in the maintenance of genome integrity is well known. However, knowledge of the regulatory role of RNAi in fungi has had to wait until the recent identification of different endogenous small RNA classes, which are generated by distinct RNAi pathways. In addition, RNAi research on new fungal models has uncovered the role of small RNAs and RNAi pathways in the regulation of diverse biological functions. In this review, we give an up-to-date overview of the different classes of small RNAs and RNAi pathways in fungi and their roles in the defense of genome integrity and regulation of fungal physiology and development, as well as in the interaction of fungi with biotic and abiotic environments.

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems.

Zymomonas mobilis has emerged as a promising candidate for production of high-value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr-like type IV restriction modification (RM) system, a type I-F CRISPR system, a chromosomal type I RM system (hsdMSc), and a previously uncharacterized type I RM system, located on an endogenous plasmid (hsdRMSp). The DNA recognition motif of HsdRMSp was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMSc and hsdRMSp systems to that of the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMSc and HsdRMSp recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction-negative strains that we constructed and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. IMPORTANCE Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study, we developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identifying a previously unknown type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis.

Evidence of repeated horizontal transfer of sterol C-5 desaturase encoding genes among dikarya fungi.

Gene duplication and horizontal gene transfer (HGT) are two important but different forces for adaptive genome evolution. In eukaryotic organisms, gene duplication is considered to play a more important evolutionary role than HGT. However, certain fungal lineages have developed highly efficient mechanisms that avoid the occurrence of duplicated gene sequences within their genomes. While these mechanisms likely originated as a defense against harmful mobile genetic elements, they come with an evolutionary cost. A prominent example for a genome defense system is the RIP mechanism of the ascomycete fungus Neurospora crassa, which efficiently prevents sequence duplication within the genome and functional redundancy of the subsequent paralogs. Despite this tight control, the fungus possesses two functionally redundant sterol C-5 desaturase enzymes, ERG-10a and ERG-10b, that catalyze the same step during ergosterol biosynthesis. In this study, we addressed this conundrum by phylogenetic analysis of the two proteins and supporting topology tests. We obtained evidence that a primary HGT of a sterol C-5 desaturase gene from Tremellales (an order of Basidiomycota) into a representative of the Pezizomycotina (a subphylum of Ascomycota) is the origin of the ERG-10b sequence. The reconstructed phylogenies suggest that this HGT event was followed by multiple HGT events among other members of the Pezizomycotina, thereby generating a diverse group with members in the four classes Sordariomycetes, Xylonomycetes, Eurotiomycetes and Dothideomycetes, which all harbor the second sterol C-5 desaturase or maintained in some cases only the ERG-10b version of this enzyme. These results furnish an example for a gene present in numerous ascomycetous fungi but primarily acquired by an ancestral HGT event from another fungal phylum. Furthermore, these data indicate that HGT represents one mechanism to generate functional redundancy in organisms with a strict avoidance of gene duplications.

A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation.

The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium isolates are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as clade A and clade B exist within E. faecium MDR E. faecium isolates belong to clade A, while clade B consists of drug-susceptible fecal commensal E. faecium isolates. Isolates from clade A are further grouped into two subclades, clades A1 and A2. In general, clade A1 isolates are hospital-epidemic isolates, whereas clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized a bioinformatics analysis coupled with second-generation and third-generation deep-sequencing platforms to characterize the methylomes of two representative E. faecium strains, one from clade A1 and one from clade B. We identified a type I R-M system that is clade A1 specific, is active for DNA methylation, and significantly reduces the transformability of clade A1 E. faecium Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M systems contribute to E. faecium subspecies separation.IMPORTANCEEnterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of this work was to determine whether restriction-modification (R-M) systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium isolates.

RNA-induced initiation of transcriptional silencing (RITS) complex structure and function.

Heterochromatic regions of the genome are epigenetically regulated to maintain a heritable '"silent state"'. In fission yeast and other organisms, epigenetic silencing is guided by nascent transcripts, which are targeted by the RNA interference pathway. The key effector complex of the RNA interference pathway consists of small interfering RNA molecules (siRNAs) associated with Argonaute, assembled into the RNA-induced transcriptional silencing (RITS) complex. This review focuses on our current understanding of how RITS promotes heterochromatin formation, and in particular on the role of Argonaute-containing complexes in many other functions such as quelling, release of RNA polymerases, cellular quiescence and genome defense.

Transposable elements: genome innovation, chromosome diversity, and centromere conflict.

Although it was nearly 70 years ago when transposable elements (TEs) were first discovered "jumping" from one genomic location to another, TEs are now recognized as contributors to genomic innovations as well as genome instability across a wide variety of species. In this review, we illustrate the ways in which active TEs, specifically retroelements, can create novel chromosome rearrangements and impact gene expression, leading to disease in some cases and species-specific diversity in others. We explore the ways in which eukaryotic genomes have evolved defense mechanisms to temper TE activity and the ways in which TEs continue to influence genome structure despite being rendered transpositionally inactive. Finally, we focus on the role of TEs in the establishment, maintenance, and stabilization of critical, yet rapidly evolving, chromosome features: eukaryotic centromeres. Across centromeres, specific types of TEs participate in genomic conflict, a balancing act wherein they are actively inserting into centromeric domains yet are harnessed for the recruitment of centromeric histones and potentially new centromere formation.

Recognizing the enemy within: licensing RNA-guided genome defense.

How do cells distinguish normal genes from transposons? Although much has been learned about RNAi-related RNA silencing pathways responsible for genome defense, this fundamental question remains. The literature points to several classes of mechanisms. In some cases, double-stranded RNA (dsRNA) structures produced by transposon inverted repeats or antisense integration trigger endogenous small interfering RNA (siRNA) biogenesis. In other instances, DNA features associated with transposons--such as their unusual copy number, chromosomal arrangement, and/or chromatin environment--license RNA silencing. Finally, recent studies have identified improper transcript processing events, such as stalled pre-mRNA splicing, as signals for siRNA production. Thus, the suboptimal gene expression properties of selfish elements can enable their identification by RNA silencing pathways.

Recurrent Gene Duplication Diversifies Genome Defense Repertoire in Drosophila.

Transposable elements (TEs) comprise large fractions of many eukaryotic genomes and imperil host genome integrity. The host genome combats these challenges by encoding proteins that silence TE activity. Both the introduction of new TEs via horizontal transfer and TE sequence evolution requires constant innovation of host-encoded TE silencing machinery to keep pace with TEs. One form of host innovation is the adaptation of existing, single-copy host genes. Indeed, host suppressors of TE replication often harbor signatures of positive selection. Such signatures are especially evident in genes encoding the piwi-interacting-RNA pathway of gene silencing, for example, the female germline-restricted TE silencer, HP1D/Rhino Host genomes can also innovate via gene duplication and divergence. However, the importance of gene family expansions, contractions, and gene turnover to host genome defense has been largely unexplored. Here, we functionally characterize Oxpecker, a young, tandem duplicate gene of HP1D/rhino We demonstrate that Oxpecker supports female fertility in Drosophila melanogaster and silences several TE families that are incompletely silenced by HP1D/Rhino in the female germline. We further show that, like Oxpecker, at least ten additional, structurally diverse, HP1D/rhino-derived daughter and "granddaughter" genes emerged during a short 15-million year period of Drosophila evolution. These young paralogs are transcribed primarily in germline tissues, where the genetic conflict between host genomes and TEs plays out. Our findings suggest that gene family expansion is an underappreciated yet potent evolutionary mechanism of genome defense diversification.

Biology, dynamics, and applications of transposable elements in basidiomycete fungi.

The phylum Basidiomycota includes filamentous fungi and yeast species with different ecological and genomic characteristics. Transposable elements (TEs) are abundant components of most eukaryotic genomes, and their transition from being genomic parasites to key drivers of genomic architecture, functionality, and evolution is a subject receiving much attention. In light of the abundant genomic information released during the last decade, the aims of this mini-review are to discuss the dynamics and impact of TEs in basidiomycete fungi. To do this, we surveyed and explored data from 75 genomes, which encompass the phylogenetic diversity of the phylum Basidiomycota. We describe annotation approaches and analyze TE distribution in the context of species phylogeny and genome size. Further, we review the most relevant literature about the role of TEs in species lifestyle, their impact on genome architecture and functionality, and the defense mechanisms evolved to control their proliferation. Finally, we discuss potential applications of TEs that can drive future innovations in fungal research.

Evolutionary interaction between W/Y chromosome and transposable elements.

The W/Y chromosome is unique among chromosomes as it does not recombine in its mature form. The main side effect of cessation of recombination is evolutionary instability and degeneration of the W/Y chromosome, or frequent W/Y chromosome turnovers. Another important feature of W/Y chromosome degeneration is transposable element (TEs) accumulation. Transposon accumulation has been confirmed for all W/Y chromosomes that have been sequenced so far. Models of W/Y chromosome instability include the assemblage of deleterious mutations in protein coding genes, but do not include the influence of transposable elements that are accumulated gradually in the non-recombining genome. The multiple roles of genomic TEs, and the interactions between retrotransposons and genome defense proteins are currently being studied intensively. Small RNAs originating from retrotransposon transcripts appear to be, in some cases, the only mediators of W/Y chromosome function. Based on the review of the most recent publications, we present knowledge on W/Y evolution in relation to retrotransposable element accumulation.

Evolutionary dynamics of the LTR-retrotransposon crapaud in the Podospora anserina species complex and the interaction with repeat-induced point mutations.

BACKGROUND: The genome of the filamentous ascomycete Podospora anserina shows a relatively high abundance of retrotransposons compared to other interspersed repeats. The LTR-retrotransposon family crapaud is particularly abundant in the genome, and consists of multiple diverged sequence variations specifically localized in the 5' half of both long terminal repeats (LTRs). P. anserina is part of a recently diverged species-complex, which makes the system ideal to classify the crapaud family based on the observed LTR variation and to study the evolutionary dynamics, such as the diversification and bursts of the elements over recent evolutionary time. RESULTS: We developed a sequence similarity network approach to classify the crapaud repeats of seven genomes representing the P. anserina species complex into 14 subfamilies. This method does not utilize a consensus sequence, but instead it connects any copies that share enough sequence similarity over a set sequence coverage. Based on phylogenetic analyses, we found that the crapaud repeats likely diversified in the ancestor of the complex and have had activity at different time points for different subfamilies. Furthermore, while we hypothesized that the evolution into multiple subfamilies could have been a direct effect of escaping the genome defense system of repeat induced point mutations, we found this not to be the case. CONCLUSIONS: Our study contributes to the development of methods to classify transposable elements in fungi, and also highlights the intricate patterns of retrotransposon evolution over short timescales and under high mutational load caused by nucleotide-altering genome defense.

The spliceosome as a transposon sensor.

The ability to distinguish self from non-self nucleic acids enables eukaryotes to suppress mobile elements and maintain genome integrity. In organisms from protist to human, this function is performed by RNA silencing pathways. There have been major advances in our understanding of the RNA silencing machinery, but the mechanisms by which these pathways distinguish self from non-self remain unclear. Recent studies in the yeast C. neoformans indicate that transposon-derived transcripts encode suboptimal introns and tend to stall in spliceosomes, which promotes the biogenesis of siRNA that targets these transcripts. These findings identify gene expression signal strength as a metric by which a foreign element can be distinguished from a host gene, and reveal a new function for introns and the spliceosome in genome defense. Anticipating that these principles may apply to RNA silencing in other systems, we discuss strong hints in the literature suggesting that the spliceosome may guide small RNA biogenesis in the siRNA and piRNA pathways of plants and animals.

A DEAD-box RNA helicase mediates meiotic silencing by unpaired DNA.

During the sexual phase of Neurospora crassa, unpaired genes are subject to a silencing mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD targets the transcripts of an unpaired gene and utilizes typical RNA interference factors for its process. Using a reverse genetic screen, we have identified a meiotic silencing gene called sad-9, which encodes a DEAD-box RNA helicase. While not essential for vegetative growth, SAD-9 plays a crucial role in both sexual development and MSUD. Our results suggest that SAD-9, with the help of the SAD-2 scaffold protein, recruits the SMS-2 Argonaute to the perinuclear region, the center of MSUD activity.

Retrocopying expands the functional repertoire of APOBEC3 antiviral proteins in primates.

Host-virus arms races are inherently asymmetric; viruses evolve much more rapidly than host genomes. Thus, there is high interest in discovering mechanisms by which host genomes keep pace with rapidly evolving viruses. One family of restriction factors, the APOBEC3 (A3) cytidine deaminases, has undergone positive selection and expansion via segmental gene duplication and recombination. Here, we show that new copies of A3 genes have also been created in primates by reverse transcriptase-encoding elements like LINE-1 or endogenous retroviruses via a process termed retrocopying. First, we discovered that all simian primate genomes retain the remnants of an ancient A3 retrocopy: A3I. Furthermore, we found that some New World monkeys encode up to ten additional APOBEC3G (A3G) retrocopies. Some of these A3G retrocopies are transcribed in a variety of tissues and able to restrict retroviruses. Our findings suggest that host genomes co-opt retroelement activity in the germline to create new host restriction factors as another means to keep pace with the rapid evolution of viruses. (163).

Analysis of Repeat Induced Point (RIP) Mutations in Leptosphaeria maculans Indicates Variability in the RIP Process Between Fungal Species.

Gene duplication contributes to evolutionary potential, yet many duplications in a genome arise from the activity of "selfish" genetic elements such as transposable elements. Fungi have a number of mechanisms by which they limit the expansion of transposons, including Repeat Induced Point mutation (RIP). RIP has been best characterized in the Sordariomycete Neurospora crassa, wherein duplicated DNA regions are recognized after cell fusion, but before nuclear fusion during the sexual cycle, and then mutated. While "signatures" of RIP appear in the genome sequences of many fungi, the species most distant from N. crassa in which the process has been experimentally demonstrated to occur is the Dothideomycete Leptosphaeria maculans In the current study, we show that similar to N. crassa, nonlinked duplications can trigger RIP; however, the frequency of the generated RIP mutations is extremely low in L maculans (< 0.1%) and requires a large duplication to initiate RIP, and that multiple premeiotic mitoses are involved in the RIP process. However, a single sexual cycle leads to the generation of progeny with unique haplotypes, despite progeny pairs being generated from mitosis. We hypothesize that these different haplotypes may be the result of the deamination process occurring post karyogamy, leading to unique mutations within each of the progeny pairs. These findings indicate that the RIP process, while common to many fungi, differs between fungi and that this impacts on the fate of duplicated DNA.

The protective function of noncoding DNA in genome defense of eukaryotic male germ cells.

Peripheral and abundant noncoding DNA has been hypothesized to protect the genome and the central protein-coding sequences against DNA damage in somatic genome. In the cytosol, invading exogenous nucleic acids may first be deactivated by small RNAs encoded by noncoding DNA via mechanisms similar to the prokaryotic CRISPR-Cas system. In the nucleus, the radicals generated by radiation in the cytosol, radiation energy and invading exogenous nucleic acids are absorbed, blocked and/or reduced by peripheral heterochromatin, and damaged DNA in heterochromatin is removed and excluded from the nucleus to the cytoplasm through nuclear pore complexes. To further strengthen the hypothesis, this review summarizes the experimental evidence supporting the protective function of noncoding DNA in the genome of male germ cells. Based on these data, this review provides evidence supporting the protective role of noncoding DNA in the genome defense of sperm genome through similar mechanisms to those of the somatic genome.