Evolution of a Restriction Factor by Domestication of a Yeast Retrotransposon.

Transposable elements drive genome evolution in all branches of life. Transposable element insertions are often deleterious to their hosts and necessitate evolution of control mechanisms to limit their spread. The long terminal repeat retrotransposon Ty1 prime (Ty1'), a subfamily of the Ty1 family, is present in many Saccharomyces cerevisiae strains, but little is known about what controls its copy number. Here, we provide evidence that a novel gene from an exapted Ty1' sequence, domesticated restriction of Ty1' relic 2 (DRT2), encodes a restriction factor that inhibits Ty1' movement. DRT2 arose through domestication of a Ty1' GAG gene and contains the C-terminal domain of capsid, which in the related Ty1 canonical subfamily functions as a self-encoded restriction factor. Bioinformatic analysis reveals the widespread nature of DRT2, its evolutionary history, and pronounced structural variation at the Ty1' relic 2 locus. Ty1' retromobility analyses demonstrate DRT2 restriction factor functionality, and northern blot and RNA-seq analysis indicate that DRT2 is transcribed in multiple strains. Velocity cosedimentation profiles indicate an association between Drt2 and Ty1' virus-like particles or assembly complexes. Chimeric Ty1' elements containing DRT2 retain retromobility, suggesting an ancestral role of productive Gag C-terminal domain of capsid functionality is present in the sequence. Unlike Ty1 canonical, Ty1' retromobility increases with copy number, suggesting that C-terminal domain of capsid-based restriction is not limited to the Ty1 canonical subfamily self-encoded restriction factor and drove the endogenization of DRT2. The discovery of an exapted Ty1' restriction factor provides insight into the evolution of the Ty1 family, evolutionary hot-spots, and host-transposable element interactions.

Reproducible evaluation of transposable element detectors with McClintock 2 guides accurate inference of Ty insertion patterns in yeast.

BACKGROUND: Many computational methods have been developed to detect non-reference transposable element (TE) insertions using short-read whole genome sequencing data. The diversity and complexity of such methods often present challenges to new users seeking to reproducibly install, execute, or evaluate multiple TE insertion detectors. RESULTS: We previously developed the McClintock meta-pipeline to facilitate the installation, execution, and evaluation of six first-generation short-read TE detectors. Here, we report a completely re-implemented version of McClintock written in Python using Snakemake and Conda that improves its installation, error handling, speed, stability, and extensibility. McClintock 2 now includes 12 short-read TE detectors, auxiliary pre-processing and analysis modules, interactive HTML reports, and a simulation framework to reproducibly evaluate the accuracy of component TE detectors. When applied to the model microbial eukaryote Saccharomyces cerevisiae, we find substantial variation in the ability of McClintock 2 components to identify the precise locations of non-reference TE insertions, with RelocaTE2 showing the highest recall and precision in simulated data. We find that RelocaTE2, TEMP, TEMP2 and TEBreak provide consistent estimates of [Formula: see text]50 non-reference TE insertions per strain and that Ty2 has the highest number of non-reference TE insertions in a species-wide panel of [Formula: see text]1000 yeast genomes. Finally, we show that best-in-class predictors for yeast applied to resequencing data have sufficient resolution to reveal a dyad pattern of integration in nucleosome-bound regions upstream of yeast tRNA genes for Ty1, Ty2, and Ty4, allowing us to extend knowledge about fine-scale target preferences revealed previously for experimentally-induced Ty1 insertions to spontaneous insertions for other copia-superfamily retrotransposons in yeast. CONCLUSION: McClintock ( https://github.com/bergmanlab/mcclintock/ ) provides a user-friendly pipeline for the identification of TEs in short-read WGS data using multiple TE detectors, which should benefit researchers studying TE insertion variation in a wide range of different organisms. Application of the improved McClintock system to simulated and empirical yeast genome data reveals best-in-class methods and novel biological insights for one of the most widely-studied model eukaryotes and provides a paradigm for evaluating and selecting non-reference TE detectors in other species.

An interchangeable prion-like domain is required for Ty1 retrotransposition.

Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report an intrinsically disordered N-terminal prion-like domain (PrLD) within Gag that is required for transposition. This domain contains amino acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wild type to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays. Our work also invites study into the prevalence of PrLDs in additional mobile elements.

An interchangeable prion-like domain is required for Ty1 retrotransposition.

Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well-understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report a novel intrinsically disordered N-terminal pr ion-like d omain (PrLD) within Gag that is required for transposition. This domain contains amino-acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wildtype to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays, and host modulators developed to study Ty1 retromobility. Our work invites study into the prevalence of PrLDs in additional mobile elements. Significance: Retrovirus-like retrotransposons help shape the genome evolution of their hosts and replicate within cytoplasmic particles. How their building blocks associate and assemble within the cell is poorly understood. Here, we report a novel pr ion-like d omain (PrLD) in the budding yeast retrotransposon Ty1 Gag protein that builds virus-like particles. The PrLD has similar sequence properties to prions and disordered protein domains that can drive the formation of assemblies that range from liquid to solid. We demonstrate that the Ty1 PrLD can function as a prion and that certain prion sequences can replace the PrLD and support Ty1 transposition. This interchangeable system is an effective platform to study additional disordered sequences in living cells.

Reproducible evaluation of transposable element detectors with McClintock 2 guides accurate inference of Ty insertion patterns in yeast.

BACKGROUND: Many computational methods have been developed to detect non-reference transposable element (TE) insertions using short-read whole genome sequencing data. The diversity and complexity of such methods often present challenges to new users seeking to reproducibly install, execute, or evaluate multiple TE insertion detectors. RESULTS: We previously developed the McClintock meta-pipeline to facilitate the installation, execution, and evaluation of six first-generation short-read TE detectors. Here, we report a completely re-implemented version of McClintock written in Python using Snakemake and Conda that improves its installation, error handling, speed, stability, and extensibility. McClintock 2 now includes 12 short-read TE detectors, auxiliary pre-processing and analysis modules, interactive HTML reports, and a simulation framework to reproducibly evaluate the accuracy of component TE detectors. When applied to the model microbial eukaryote Saccharomyces cerevisiae, we find substantial variation in the ability of McClintock 2 components to identify the precise locations of non-reference TE insertions, with RelocaTE2 showing the highest recall and precision in simulated data. We find that RelocaTE2, TEMP, TEMP2 and TEBreak provide a consistent and biologically meaningful view of non-reference TE insertions in a species-wide panel of ∻1000 yeast genomes, as evaluated by coverage-based abundance estimates and expected patterns of tRNA promoter targeting. Finally, we show that best-in-class predictors for yeast have sufficient resolution to reveal a dyad pattern of integration in nucleosome-bound regions upstream of yeast tRNA genes for Ty1, Ty2, and Ty4, allowing us to extend knowledge about fine-scale target preferences first revealed experimentally for Ty1 to natural insertions and related copia-superfamily retrotransposons in yeast. CONCLUSION: McClintock (https://github.com/bergmanlab/mcclintock/) provides a user-friendly pipeline for the identification of TEs in short-read WGS data using multiple TE detectors, which should benefit researchers studying TE insertion variation in a wide range of different organisms. Application of the improved McClintock system to simulated and empirical yeast genome data reveals best-in-class methods and novel biological insights for one of the most widely-studied model eukaryotes and provides a paradigm for evaluating and selecting non-reference TE detectors for other species.

Horizontal transfer and recombination fuel Ty4 retrotransposon evolution in Saccharomyces.

Horizontal transposon transfer (HTT) plays an important role in the evolution of eukaryotic genomes, however the detailed evolutionary history and impact of most HTT events remain to be elucidated. To better understand the process of HTT in closely-related microbial eukaryotes, we studied Ty4 retrotransposon subfamily content and sequence evolution across the genus Saccharomyces using short- and long-read whole genome sequence data, including new PacBio genome assemblies for two S. mikatae strains. We find evidence for multiple independent HTT events introducing the Tsu4 subfamily into specific lineages of S. paradoxus, S. cerevisiae, S. eubayanus, S. kudriavzevii and the ancestor of the S. mikatae/S. jurei species pair. In both S. mikatae and S. kudriavzevii, we identified novel Ty4 clades that were independently generated through recombination between resident and horizontally-transferred subfamilies. Our results reveal that recurrent HTT and lineage-specific extinction events lead to a complex pattern of Ty4 subfamily content across the genus Saccharomyces. Moreover, our results demonstrate how HTT can lead to coexistence of related retrotransposon subfamilies in the same genome that can fuel evolution of new retrotransposon clades via recombination.

Cell Compartment-Specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome.

The structural transitions RNAs undergo during trafficking are not well understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and those synthesized in vitro, we detected Ty1 gRNA structural alterations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, whereas the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA-RNA interactions required for retrotransposition.

Genome Assembly of the Ty1-Less Saccharomyces paradoxus Strain DG1768.

Here, we report an essentially complete genome assembly for the Ty1-less Saccharomyces paradoxus strain DG1768 (derivative of strain 337) based on PacBio and Illumina shotgun sequence data. We also document the genetic alterations that make this yeast strain a key resource for Ty1 mobility studies.

Long-Read Genome Assembly of Saccharomyces uvarum Strain CBS 7001.

Here, we report a long-read genome assembly for Saccharomyces uvarum strain CBS 7001 based on PacBio whole-genome shotgun sequence data. Our assembly provides an improved reference genome for an important yeast in the Saccharomyces sensu stricto clade.

Structure of a Ty1 restriction factor reveals the molecular basis of transposition copy number control.

Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism.

RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein.

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

In vivo structure of the Ty1 retrotransposon RNA genome.

Long terminal repeat (LTR)-retrotransposons constitute a significant part of eukaryotic genomes and influence their function and evolution. Like other RNA viruses, LTR-retrotransposons efficiently utilize their RNA genome to interact with host cell machinery during replication. Here, we provide the first genome-wide RNA secondary structure model for a LTR-retrotransposon in living cells. Using SHAPE probing, we explore the secondary structure of the yeast Ty1 retrotransposon RNA genome in its native in vivo state and under defined in vitro conditions. Comparative analyses reveal the strong impact of the cellular environment on folding of Ty1 RNA. In vivo, Ty1 genome RNA is significantly less structured and more dynamic but retains specific well-structured regions harboring functional cis-acting sequences. Ribosomes participate in the unfolding and remodeling of Ty1 RNA, and inhibition of translation initiation stabilizes Ty1 RNA structure. Together, our findings support the dual role of Ty1 genomic RNA as a template for protein synthesis and reverse transcription. This study also contributes to understanding how a complex multifunctional RNA genome folds in vivo, and strengthens the need for studying RNA structure in its natural cellular context.

Evolution of Ty1 copy number control in yeast by horizontal transfer and recombination.

Transposable elements constitute a large fraction of most eukaryotic genomes. Insertion of mobile DNA sequences typically has deleterious effects on host fitness, and thus diverse mechanisms have evolved to control mobile element proliferation. Mobility of the Ty1 retrotransposon in Saccharomyces yeasts is regulated by copy number control (CNC) mediated by a self-encoded restriction factor derived from the Ty1 gag capsid gene that inhibits virus-like particle function. Here, we survey a panel of wild and human-associated strains of S. cerevisiae and S. paradoxus to investigate how genomic Ty1 content influences variation in Ty1 mobility. We observe high levels of mobility for a tester element with a gag sequence from the canonical Ty1 subfamily in permissive strains that either lack full-length Ty1 elements or only contain full-length copies of the Ty1' subfamily that have a divergent gag sequence. In contrast, low levels of canonical Ty1 mobility are observed in restrictive strains carrying full-length Ty1 elements containing a canonical gag sequence. Phylogenomic analysis of full-length Ty1 elements revealed that Ty1' is the ancestral subfamily present in wild strains of S. cerevisiae, and that canonical Ty1 in S. cerevisiae is a derived subfamily that acquired gag from S. paradoxus by horizontal transfer and recombination. Our results provide evidence that variation in the ability of S. cerevisiae and S. paradoxus strains to repress canonical Ty1 transposition via CNC is regulated by the genomic content of different Ty1 subfamilies, and that self-encoded forms of transposon control can spread across species boundaries by horizontal transfer.

Retroviral-like determinants and functions required for dimerization of Ty1 retrotransposon RNA.

During replication of long terminal repeat (LTR)-retrotransposons, their proteins and genome (g) RNA assemble into virus-like particles (VLPs) that are not infectious but functionally related to retroviral virions. Both virions and VLPs contain gRNA in a dimeric form, but contrary to retroviruses, little is known about how gRNA dimerization and packaging occurs in LTR-retrotransposons. The LTR-retrotransposon Ty1 from Saccharomyces cerevisiae is an informative model for studying LTR-retrotransposon and retrovirus replication. Using structural, mutational and functional analyses, we explored dimerization of Ty1 genomic RNA. We provide direct evidence that interactions of self-complementary PAL1 and PAL2 palindromic sequences localized within the 5'UTR are essential for Ty1 gRNA dimer formation. Mutations disrupting PAL1-PAL2 complementarity restricted RNA dimerization in vitro and Ty1 mobility in vivo. Although dimer formation and mobility of these mutants was inhibited, our work suggests that Ty1 RNA can dimerize via alternative contact points. In contrast to previous studies, we cannot confirm a role for PAL3, tRNAiMet as well as recently proposed initial kissing-loop interactions in dimer formation. Our data also supports the critical role of Ty1 Gag in RNA dimerization. Mature Ty1 Gag binds in the proximity of sequences involved in RNA dimerization and tRNAiMet annealing, but the 5' pseudoknot in Ty1 RNA may constitute a preferred Gag-binding site. Taken together, these results expand our understanding of genome dimerization and packaging strategies utilized by LTR-retroelements.

Ribosome Biogenesis Modulates Ty1 Copy Number Control in Saccharomyces cerevisiae.

Transposons can impact the host genome by altering gene expression and participating in chromosome rearrangements. Therefore, organisms evolved different ways to minimize the level of transposition. In Saccharomyces cerevisiae and its close relative S. paradoxus, Ty1 copy number control (CNC) is mediated by the self-encoded restriction factor p22, which is derived from the GAG capsid gene and inhibits virus-like particle (VLP) assembly and function. Based on secondary screens of Ty1 cofactors, we identified LOC1, a RNA localization/ribosome biogenesis gene that affects Ty1 mobility predominantly in strains harboring Ty1 elements. Ribosomal protein mutants rps0bΔ and rpl7aΔ displayed similar CNC-specific phenotypes as loc1Δ, suggesting that ribosome biogenesis is critical for CNC. The level of Ty1 mRNA and Ty1 internal (Ty1i) transcripts encoding p22 was altered in these mutants, and displayed a trend where the level of Ty1i RNA increased relative to full-length Ty1 mRNA. The level of p22 increased in these mutants, and the half-life of p22 also increased in a loc1Δ mutant. Transcriptomic analyses revealed small changes in the level of Ty1 transcripts or efficiency of translation initiation in a loc1Δ mutant. Importantly, a loc1Δ mutant had defects in assembly of Gag complexes and packaging Ty1 RNA. Our results indicate that defective ribosome biogenesis enhances CNC by increasing the level of p22, and raise the possibility for versatile links between VLP assembly, its cytoplasmic environment, and a novel stress response.

Structure of Ty1 Internally Initiated RNA Influences Restriction Factor Expression.

The long-terminal repeat retrotransposon Ty1 is the most abundant mobile genetic element in many Saccharomyces cerevisiae isolates. Ty1 retrotransposons contribute to the genetic diversity of host cells, but they can also act as an insertional mutagen and cause genetic instability. Interestingly, retrotransposition occurs at a low level despite a high level of Ty1 RNA, even though S. cerevisiae lacks the intrinsic defense mechanisms that other eukaryotes use to prevent transposon movement. p22 is a recently discovered Ty1 protein that inhibits retrotransposition in a dose-dependent manner. p22 is a truncated form of Gag encoded by internally initiated Ty1i RNA that contains two closely-spaced AUG codons. Mutations of either AUG codon compromise p22 translation. We found that both AUG codons were utilized and that translation efficiency depended on the Ty1i RNA structure. Structural features that stimulated p22 translation were context dependent and present only in Ty1i RNA. Destabilization of the 5' untranslated region (5' UTR) of Ty1i RNA decreased the p22 level, both in vitro and in vivo. Our data suggest that protein factors such as Gag could contribute to the stability and translational activity of Ty1i RNA through specific interactions with structural motifs in the RNA.

Characterizing the functions of Ty1 Gag and the Gag-derived restriction factor p22/p18.

The long terminal repeat (LTR) and non-LTR retrotransposons comprise approximately half of the human genome, and we are only beginning to understand their influence on genome function and evolution. The LTR retrotransposon Ty1 is the most abundant mobile genetic element in the S. cerevisiae reference genome. Ty1 replicates via an RNA intermediate and shares several important structural and functional characteristics with retroviruses. However, unlike retroviruses Ty1 retrotransposition is not infectious. Retrotransposons integrations can cause mutations and genome instability. Despite the fact that S. cerevisiae lacks eukaryotic defense mechanisms such as RNAi, they maintain a relatively low copy number of the Ty1 retrotransposon in their genomes. A novel restriction factor derived from the C-terminal half of Gag (p22/p18) and encoded by internally initiated transcript inhibits retrotransposition in a dose-dependent manner. Therefore, Ty1 evolved a specific GAG organization and expression strategy to produce products both essential and antagonistic for retrotransposon movement. In this commentary we discuss our recent research aimed at defining steps of Ty1 replication influenced by p22/p18 with particular emphasis on the nucleic acid chaperone functions carried out by Gag and the restriction factor.

Ty1 escapes restriction by the self-encoded factor p22 through mutations in capsid.

Ty1 is a long terminal repeat (LTR) retrotransposon belonging to the Ty1/copia family and is present in up to 32 full-length copies in Saccharomyces. Like retroviruses, Ty1 contains GAG and POL genes, LTRs, and replicates via an RNA intermediate within a virus-like particle (VLP). Although Ty1 retrotransposition is not infectious, uncontrolled replication can lead to detrimental effects on the host genome, including insertional mutagenesis and chromosomal rearrangements. Ty1 copy number control (CNC) limits replication and is mediated through a self-encoded protein called p22. p22 is translated from a subgenomic Ty1 RNA and encodes an amino-truncated version of the Gag protein. We highlight a recent study identifying Ty1 Gag, which comprises the VLP capsid and provides nucleic acid chaperone functions, as a direct target of p22-mediated inhibition. CNC-resistant (CNCR) mutations map within predicted helical domains of Gag, including those in the Ty1/copia pfam domain Retrotran_gag_2 (formerly UBN2) and a central region we refer to as the CNCR domain. CNCR Gag forms VLPs that exclude p22, thus restoring Ty1 replication. We discuss possible mechanisms for p22 inclusion in Ty1 VLPs and compare Ty1 CNC with retroviral restriction factors targeting capsid (CA).