Population genetic analysis based on the polymorphisms mediated by transposons in the genomes of pig.

Transposable elements (TEs) mobility is capable of generating a large number of structural variants (SVs), which can have considerable potential as molecular markers for genetic analysis and molecular breeding in livestock. Our results showed that the pig genome contains mainly TE-SVs generated by short interspersed nuclear elements (51,873/76.49%), followed by long interspersed nuclear elements (11,131/16.41%), and more than 84% of the common TE-SVs (Minor allele frequency, MAF > 0.10) were validated to be polymorphic. Subsequently, we utilized the identified TE-SVs to gain insights into the population structure, resulting in clear differentiation among the three pig groups and facilitating the identification of relationships within Chinese local pig breeds. In addition, we investigated the frequencies of TEs in the gene coding regions of different pig groups and annotated the respective TE types, related genes, and functional pathways. Through genome-wide comparisons of Large White pigs and Chinese local pigs utilizing the Beijing Black pigs, we identified TE-mediated SVs associated with quantitative trait loci and observed that they were mainly involved in carcass traits and meat quality traits. Lastly, we present the first documented evidence of TE transduction in the pig genome.

Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation.

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA-RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs.

Machine learning to detect the SINEs of cancer.

We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.

A SINE-VNTR-Alu at the LRIG2 locus is associated with proximal and distal gene expression in CRISPR and population models.

SINE-VNTR-Alu (SVA) retrotransposons represent mobile regulatory elements that have the potential to influence the surrounding genome when they insert into a locus. Evolutionarily recent mobilisation has resulted in loci in the human genome where a given retrotransposon might be observed to be present or absent, termed a retrotransposon insertion polymorphism (RIP). We previously observed that an SVA RIP ~ 2 kb upstream of LRIG2 on chromosome 1, the 'LRIG2 SVA', was associated with differences in local gene expression and methylation, and that the two were correlated. Here, we have used CRISPR-mediated deletion of the LRIG2 SVA in a cell line model to validate that presence of the retrotransposon is directly affecting local expression and provide evidence that is suggestive of a modest role for the SVA in modulating nearby methylation. Additionally, in leveraging an available Hi-C dataset we observed that the LRIG2 SVA was also involved in long-range chromatin interactions with a cluster of genes ~ 300 kb away, and that expression of these genes was to varying degrees associated with dosage of the SVA in both CRISPR cell line and population models. Altogether, these data support a regulatory role for SVAs in the modulation of gene expression, with the latter potentially involving chromatin looping, consistent with the model that RIPs may contribute to interpersonal differences in transcriptional networks.

Interspecific Comparison of Orthologous Short Interspersed Elements Loci Using Whole-Genome Data.

The polymorphism of SINE-containing loci reflects the evolutionary processes that occurred both during the period before the divergence of the taxa and after it. Orthologous loci containing SINE in two or more genomes indicate the relatedness of the taxa, while different copies may have a specific set of mutations and degree of difference. Polymorphic insertion can be interpreted with a high degree of confidence as a shared derived character in the phylogenetic reconstruction of the history of the taxon. The computational comparison of the entire set of SINE-containing loci between genomes is a challenging task, and we propose to consider it in detail using the genomes of representatives of squamate reptiles (lizards) as an example. Our approach allows us to extract copies of SINE from the genomes, find pairwise orthologous loci by using flanking genomic sequences, and analyze the resulting sets of loci for the presence or absence of SINE, the degree of similarity of the flanks, and the similarity of the SINE themselves. The workflow we propose allows us to efficiently extract and analyze orthologous SINE loci for the downstream analysis, as shown in our comparison of species- and genus-level taxa in lacertid lizards.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

SINEs as Potential Expression Cassettes: Impact of Deletions and Insertions on Polyadenylation and Lifetime of B2 and Ves SINE Transcripts Generated by RNA Polymerase III.

Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should contain the AATAAA sequence as well as those called ß- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are ß- and τ-signals. Extended regions outside of ß- and τ-signals were deleted from SINEs B2 and Ves and the derived constructs were used to transfect HeLa cells in order to evaluate the relative levels of their transcripts as well as their polyadenylation efficiency. If the deleted regions affected boxes A and B, the 5'-flanking region of the U6 RNA gene with the external promoter was inserted upstream. Such substitution of the internal promoter in B2 completely restored its transcription. Almost all tested deletions/substitutions did not reduce the polyadenylation capacity of the transcripts, indicating a weak dependence of the function of ß- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between ß- and τ-signals showed an equal polyadenylation efficiency of their transcripts compared to those of constructs without the insertion. The acquired poly(A)-tails significantly increased the lifetime and thus the cellular level of such transcripts. The data obtained highlight the potential of B2 and Ves SINEs as cassettes for the expression of relatively short sequences for various applications.

Young SINEs in pig genomes impact gene regulation, genetic diversity, and complex traits.

Transposable elements (TEs) are a major source of genetic polymorphisms and play a role in chromatin architecture, gene regulatory networks, and genomic evolution. However, their functional role in pigs and contributions to complex traits are largely unknown. We created a catalog of TEs (n = 3,087,929) in pigs and found that young SINEs were predominantly silenced by histone modifications, DNA methylation, and decreased accessibility. However, some transcripts from active young SINEs showed high tissue-specificity, as confirmed by analyzing 3570 RNA-seq samples. We also detected 211,067 dimorphic SINEs in 374 individuals, including 340 population-specific ones associated with local adaptation. Mapping these dimorphic SINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes (e.g., ANK2 and VRTN) that might be mediated by TEs. Our findings highlight the important roles of young SINEs and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs.

Seq-ing the SINEs of central nervous system tumors in cerebrospinal fluid.

It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.

Extensive Independent Amplification of Platy-1 Retroposons in Tamarins, Genus Saguinus.

Platy-1 retroposons are short interspersed elements (SINEs) unique to platyrrhine primates. Discovered in the common marmoset (Callithrix jacchus) genome in 2016, these 100 bp mobile element insertions (MEIs) appeared to be novel drivers of platyrrhine evolution, with over 2200 full-length members across 62 different subfamilies, and strong evidence of ongoing proliferation in C. jacchus. Subsequent characterization of Platy-1 elements in Aotus, Saimiri and Cebus genera, suggested that the widespread mobilization detected in marmoset (family Callithrichidae) was perhaps an anomaly. Two additional Callithrichidae genomes are now available, a scaffold level genome assembly for Saguinus imperator (tamarin; SagImp_v1) and a chromosome-level assembly for Saguinus midas (Midas tamarin; ASM2_v1). Here, we report that each tamarin genome contains over 11,000 full-length Platy-1 insertions, about 1150 are shared by both Saguinus tamarins, 7511 are unique to S. imperator, and another 8187 are unique to S. midas. Roughly 325 are shared among the three callithrichids. We identified six new Platy-1 subfamilies derived from Platy-1-8, with the youngest new subfamily, Platy-1-8c_Saguinus, being the primary source of the Saguinus amplification burst. This constitutes the largest expansion of Platy-1 MEIs reported to date and the most extensive independent SINE amplification between two closely related species.

A Genome-Wide Screen for the Exonisation of Reference SINE-VNTR-Alus and Their Expression in CNS Tissues of Individuals with Amyotrophic Lateral Sclerosis.

The hominid-specific retrotransposon SINE-VNTR-Alu (SVA) is a composite element that has contributed to the genetic variation between individuals and influenced genomic structure and function. SVAs are involved in modulating gene expression and splicing patterns, altering mRNA levels and sequences, and have been associated with the development of disease. We evaluated the genome-wide effects of SVAs present in the reference genome on transcript sequence and expression in the CNS of individuals with and without the neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS). This study identified SVAs in the exons of 179 known transcripts, several of which were expressed in a tissue-specific manner, as well as 92 novel exonisation events occurring in the motor cortex. An analysis of 65 reference genome SVAs polymorphic for their presence/absence in the ALS consortium cohort did not identify any elements that were significantly associated with disease status, age at onset, and survival. However, there were transcripts, such as transferrin and HLA-A, that were differentially expressed between those with or without disease, and expression levels were associated with the genotype of proximal SVAs. This study demonstrates the functional consequences of several SVA elements altering mRNA splicing patterns and expression levels in tissues of the CNS.

Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.

SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas.

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

Maternal Immune Activation and Enriched Environments Impact B2 SINE Expression in Stress Sensitive Brain Regions of Rodent Offspring.

Early life stress (ELS) can have wide-spread neurodevelopmental effects with support accumulating for the idea that genomic mechanisms may induce lasting physiological and behavioral changes following stress exposure. Previous work found that a sub-family of transposable elements, SINEs, are repressed epigenetically after acute stress. This gives support to the concept that the mammalian genome may be regulating retrotransposon RNA expression allowing for adaptation in response to environmental challenges, such as maternal immune activation (MIA). Transposon (TE) RNAs are now thought to work at the epigenetic level and to have an adaptive response to environmental stressors. Abnormal expression of TEs has been linked to neuropsychiatric disorders like schizophrenia, which is also linked to maternal immune activation. Environmental enrichment (EE), a clinically utilized intervention, is understood to protect the brain, enhance cognitive performance, and attenuate responses to stress. This study examines the effects of MIA on offspring B2 SINE expression and further, the impact that EE, experienced throughout gestation and early life, may have in conjunction with MIA during development. Utilizing RT-PCR to quantify the expression of B2 SINE RNA in the juvenile brain of MIA exposed rat offspring, we found dysregulation of B2 SINE expression associated with MIA in the prefrontal cortex. For offspring experiencing EE, the prefrontal cortex exhibited an attenuation of the MIA response observed in standard housed animals. Here, the adaptive nature of B2 is observed and thought to be aiding in the animal's adaptation to stress. The present changes indicate a wide-spread stress-response system adaptation that impacts not only changes at the genomic level but potentially observable behavioral impacts throughout the lifespan, with possible translational relevance to psychotic disorders.

Bridging multiple dimensions: roles of transposable elements in higher-order genome regulation.

Transposable elements (TEs) such as endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs) occupy nearly half of typical mammalian genomes. Previous studies show that these parasitic elements, especially LINEs and ERVs, provide important activities promoting host germ cell and placental development, preimplantation embryogenesis, and maintenance of pluripotent stem cells. Despite being the most numerically abundant type of TEs in the genome, the consequences of SINEs on host genome regulation are less well characterized than those of ERVs and LINEs. Interestingly, recent findings reveal that SINEs recruit the key architectural protein CTCF (CCCTC-binding factor), indicating a role of these elements for 3D genome regulation. Higher-order nuclear structures are linked with important cellular functions such as gene regulation and DNA replication. SINEs and other TEs, therefore, may mediate distinct physiological processes with benefits to the host by modulating the 3D genome.

Hominoid SVA-lncRNA AK057321 targets human-specific SVA retrotransposons in SCN8A and CDK5RAP2 to initiate neuronal maturation.

SINE-VNTR-Alu (SVA) retrotransposons arose and expanded in the genome of hominoid primates concurrent with the slowing of brain maturation. We report genes with intronic SVA transposons are enriched for neurodevelopmental disease and transcribed into long non-coding SVA-lncRNAs. Human-specific SVAs in microcephaly CDK5RAP2 and epilepsy SCN8A gene introns repress their expression via transcription factor ZNF91 to delay neuronal maturation. Deleting the SVA in CDK5RAP2 initiates multi-dimensional and in SCN8A selective sodium current neuronal maturation by upregulating these genes. SVA-lncRNA AK057321 forms RNA:DNA heteroduplexes with the genomic SVAs and upregulates these genes to initiate neuronal maturation. SVA-lncRNA AK057321 also promotes species-specific cortex and cerebellum-enriched expression upregulating human genes with intronic SVAs (e.g., HTT, CHAF1B and KCNJ6) but not mouse orthologs. The diversity of neuronal genes with intronic SVAs suggest this hominoid-specific SVA transposon-based gene regulatory mechanism may act at multiple steps to specialize and achieve neoteny of the human brain.

Exact solutions of (1 + 1)-dimensional integro-differential Ito, KP hierarchy, CBS, MCBS and modified KdV-CBS equations.

The present study computes the Lie symmetries and exact solutions of some problems modeled by nonlinear partial differential equations. The (1 + 1)-dimensional integro-differential Ito, the first integro-differential KP hierarchy, the Calogero-Bogoyavlenskii-Schiff (CBS), the modified Calogero-Bogoyavlenskii-Schiff (CBS), and the modified KdV-CBS equations are some of the problems for which we want to find new exact solutions. We employ similarity variables to reduce the number of independent variables and inverse similarity transformations to obtain exact solutions to the equations under consideration. The sine-cosine method is then utilized to determine the exact solutions.

Contribution of Retrotransposons to the Pathogenesis of Type 1 Diabetes and Challenges in Analysis Methods.

Type 1 diabetes (T1D) is one of the most common chronic diseases of the endocrine system, associated with several life-threatening comorbidities. While the etiopathogenesis of T1D remains elusive, a combination of genetic susceptibility and environmental factors, such as microbial infections, are thought to be involved in the development of the disease. The prime model for studying the genetic component of T1D predisposition encompasses polymorphisms within the HLA (human leukocyte antigen) region responsible for the specificity of antigen presentation to lymphocytes. Apart from polymorphisms, genomic reorganization caused by repeat elements and endogenous viral elements (EVEs) might be involved in T1D predisposition. Such elements are human endogenous retroviruses (HERVs) and non-long terminal repeat (non-LTR) retrotransposons, including long and short interspersed nuclear elements (LINEs and SINEs). In line with their parasitic origin and selfish behaviour, retrotransposon-imposed gene regulation is a major source of genetic variation and instability in the human genome, and may represent the missing link between genetic susceptibility and environmental factors long thought to contribute to T1D onset. Autoreactive immune cell subtypes with differentially expressed retrotransposons can be identified with single-cell transcriptomics, and personalized assembled genomes can be constructed, which can then serve as a reference for predicting retrotransposon integration/restriction sites. Here we review what is known to date about retrotransposons, we discuss the involvement of viruses and retrotransposons in T1D predisposition, and finally we consider challenges in retrotransposons analysis methods.

LINE-1 Retrotransposition Assays in Embryonic Stem Cells.

The ongoing mobilization of active non-long terminal repeat (LTR) retrotransposons continues to impact the genomes of most mammals, including humans and rodents. Non-LTR retrotransposons mobilize using an intermediary RNA and a copy-and-paste mechanism termed retrotransposition. Non-LTR retrotransposons are subdivided into long and short interspersed elements (LINEs and SINEs, respectively), depending on their size and autonomy; while active class 1 LINEs (LINE-1s or L1s) encode the enzymatic machinery required to mobilize in cis, active SINEs use the enzymatic machinery of active LINE-1s to mobilize in trans. The mobilization mechanism used by LINE-1s/SINEs was exploited to develop ingenious plasmid-based retrotransposition assays in cultured cells, which typically exploit a reporter gene that can only be activated after a round of retrotransposition. Retrotransposition assays, in cis or in trans, are instrumental tools to study the biology of mammalian LINE-1s and SINEs. In fact, these and other biochemical/genetic assays were used to uncover that endogenous mammalian LINE-1s/SINEs naturally retrotranspose during early embryonic development. However, embryonic stem cells (ESCs) are typically used as a cellular model in these and other studies interrogating LINE-1/SINE expression/regulation during early embryogenesis. Thus, human and mouse ESCs represent an excellent model to understand how active retrotransposons are regulated and how their activity impacts the germline. Here, we describe robust and quantitative protocols to study human/mouse LINE-1 (in cis) and SINE (in trans) retrotransposition using (human and mice) ESCs. These protocols are designed to study the mobilization of active non-LTR retrotransposons in a cellular physiologically relevant context.

SINEs as Credible Signs to Prove Common Ancestry in the Tree of Life: A Brief Review of Pioneering Case Studies in Retroposon Systematics.

Currently, the insertions of SINEs (and other retrotransposed elements) are regarded as one of the most reliable synapomorphies in molecular systematics. The methodological mainstream of molecular systematics is the calculation of nucleotide (or amino acid) sequence divergences under a suitable substitution model. In contrast, SINE insertion analysis does not require any complex model because SINE insertions are unidirectional and irreversible. This straightforward methodology was named the "SINE method," which resolved various taxonomic issues that could not be settled by sequence comparison alone. The SINE method has challenged several traditional hypotheses proposed based on the fossil record and anatomy, prompting constructive discussions in the Evo/Devo era. Here, we review our pioneering SINE studies on salmon, cichlids, cetaceans, Afrotherian mammals, and birds. We emphasize the power of the SINE method in detecting incomplete lineage sorting by tracing the genealogy of specific genomic loci with minimal noise. Finally, in the context of the whole-genome era, we discuss how the SINE method can be applied to further our understanding of the tree of life.