SVA Elements Research Articles

9269 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development. Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

9269 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development. Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8703 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development.Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8703 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development.Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

Emerging Opportunities to Study Mobile Element Insertions and Their Source Elements in an Expanding Universe of Sequenced Human Genomes.

Three mobile element classes, namely Alu, LINE-1 (L1), and SVA elements, remain actively mobile in human genomes and continue to produce new mobile element insertions (MEIs). Historically, MEIs have been discovered and studied using several methods, including: (1) Southern blots, (2) PCR (including PCR display), and (3) the detection of MEI copies from young subfamilies. We are now entering a new phase of MEI discovery where these methods are being replaced by whole genome sequencing and bioinformatics analysis to discover novel MEIs. We expect that the universe of sequenced human genomes will continue to expand rapidly over the next several years, both with short-read and long-read technologies. These resources will provide unprecedented opportunities to discover MEIs and study their impact on human traits and diseases. They also will allow the MEI community to discover and study the source elements that produce these new MEIs, which will facilitate our ability to study source element regulation in various tissue contexts and disease states. This, in turn, will allow us to better understand MEI mutagenesis in humans and the impact of this mutagenesis on human biology.

Reference LINE-1 insertion polymorphisms correlate with Parkinson’s disease progression and differential transcript expression in the PPMI cohort

Long interspersed nuclear element-1 (LINE-1/L1) retrotransposons make up 17% of the human genome. They represent one class of transposable elements with the capacity to both mobilize autonomously and in trans via the mobilization of other elements, primarily Alu and SVA elements. Reference LINE-1 elements are, by definition, found in the reference genome, however, due to the polymorphic nature of these elements, variation for presence or absence is present within the population. We used a combination of clinical and transcriptomic data from the Parkinson’s Progression Markers Initiative (PPMI) and applied matrix expression quantitative trait loci analysis and linear mixed-effects models involving 114 clinical, biochemical and imaging data from the PPMI cohort to elucidate the role of reference LINE-1 insertion polymorphism on both gene expression genome-wide and progression of Parkinson’s disease (PD). We demonstrate that most LINE-1 insertion polymorphisms are capable of regulating gene expression, preferentially in trans, including previously identified PD risk loci. In addition, we show that 70 LINE-1 elements were associated with longitudinal changes of at least one PD progression marker, including ipsilateral count density ratio and UPDRS scores which are indicators of degeneration and severity. In conclusion, this study highlights the effect of the polymorphic nature of LINE-1 retrotransposons on gene regulation and progression of PD which underlines the importance of analyzing transposable elements within complex diseases.

Being and Becoming: The Complementarity of Creation and Evolution

Longstanding debates relating to biological evolution concern whether random events (mutations of DNA) are able to generate new functionality, and whether such proposed evolutionary mechanisms are compatible with belief in divine creation. The sequencing of genomes from multiple species has generated a flood of genomic data, so that genetic changes may be correlated with species’ phenotypes. Our genomes are modified by mutagenic agents such as retroviruses (ERVs) and transposable elements (TEs). Empirical data confirm that random accumulations of ERVs and TEs in the human genome have rewired regulatory networks in early embryos (ERV-like MaLR elements), embryonic stem cells (ERV-H), and primordial germ cells (ERV-K). Altered regulation of gene activity in neural cells has been evinced for a class of TEs called SVA elements. Random, stochastic events in the context of natural laws that are hospitable to life may indeed generate new genetic information. Christians may see such phenomena as aspects of a freely operating and fruitful creation. Acceptance of biological evolution and the role of randomness in an anthropic cosmos are indeed compatible with the biblical concept of creation—that the whole system is ordained, ordered, and sustained by a purposeful and self-revealing God.

Human Retrotransposons and Effective Computational Detection Methods for Next-Generation Sequencing Data.

Transposable elements (TEs) are classified into two classes according to their mobilization mechanism. Compared to DNA transposons that move by the “cut and paste” mechanism, retrotransposons mobilize via the “copy and paste” method. They have been an essential research topic because some of the active elements, such as Long interspersed element 1 (LINE-1), Alu, and SVA elements, have contributed to the genetic diversity of primates beyond humans. In addition, they can cause genetic disorders by altering gene expression and generating structural variations (SVs). The development and rapid technological advances in next-generation sequencing (NGS) have led to new perspectives on detecting retrotransposon-mediated SVs, especially insertions. Moreover, various computational methods have been developed based on NGS data to precisely detect the insertions and deletions in the human genome. Therefore, this review discusses details about the recently studied and utilized NGS technologies and the effective computational approaches for discovering retrotransposons through it. The final part covers a diverse range of computational methods for detecting retrotransposon insertions with human NGS data. This review will give researchers insights into understanding the TEs and how to investigate them and find connections with research interests.

A Map of 3' DNA Transduction Variants Mediated by Non-LTR Retroelements on 3202 Human Genomes.

Simple SummaryDuring the transcription of non-LTR retroelements, such as LINEs and SVAs, the transcriptional termination signal at the 3′ end might be ignored by RNA polymerase. As a result, the transcription terminates at another downstream signal, creating a chimeric transcriptional readthrough. Termed 3′ DNA transduction, this process copies the 3′ flanking region along with the retroelement sequence to a new genomic locus, which influences the structure of the genome and occasionally possesses a functional impact. To discover putative non-LTR retroelement-driven 3′ DNA transductions, we analyzed the new dataset (n = 3202) of the 1000 Genomes Project. Our results indicate that 3′ transduction derived by non-LTR retroelements is a relatively common phenomenon in the human genome and that their discovery needs to be more appreciated in genome projects.As one of the major structural constituents, mobile elements comprise more than half of the human genome, among which Alu, L1, and SVA elements are still active and continue to generate new offspring. One of the major characteristics of L1 and SVA elements is their ability to co-mobilize adjacent downstream sequences to new loci in a process called 3′ DNA transduction. Transductions influence the structure and content of the genome in different ways, such as increasing genome variation, exon shuffling, and gene duplication. Moreover, given their mutagenicity capability, 3′ transductions are often involved in tumorigenesis or in the development of some diseases. In this study, we analyzed 3202 genomes sequenced at high coverage by the New York Genome Center to catalog and characterize putative 3′ transduced segments mediated by L1s and SVAs. Here, we present a genome-wide map of inter/intrachromosomal 3′ transduction variants, including their genomic and functional location, length, progenitor location, and allelic frequency across 26 populations. In total, we identified 7103 polymorphic L1s and 3040 polymorphic SVAs. Of these, 268 and 162 variants were annotated as high-confidence L1 and SVA 3′ transductions, respectively, with lengths that ranged from 7 to 997 nucleotides. We found specific loci within chromosomes X, 6, 7, and 6_GL000253v2_alt as master L1s and SVAs that had yielded more transductions, among others. Together, our results demonstrate the dynamic nature of transduction events within the genome and among individuals and their contribution to the structural variations of the human genome.

Accelerated Variability of Human Genes and Transportable Elements; Genesis of Network

In this review the authors address the issues related to the evolution of human. A human differs from all other species in that she acts according to a plan, or an idea she has chosen. The discovery of HAR (human accelerated region) showed that evolutionarily new regulatory regions play an important role in the functioning and development of the human brain. In Homo sapiens, conserved sequences in this area underwent numerous single nucleotide substitutions. In the five selected HARs, substitution rates were 26 times higher than those for chimpanzees showing 63 extremely fast-paced regions for H. sapiens. Human genes that regulate the development of the nervous system during evolution underwent positive selection mainly within their non-coding sequences. 92% of the detected HARs are located in intergenic regions and introns and therefore are regulatory sequences, such as enhancers. Only 2% of our genome consists of genes encoding a protein, and the remaining 98% encode regulatory elements that control gene expression in different tissues. Eukaryotic genomes contain thousands to millions of copies of transportable elements (TE). Authors believe that evolution is driven by the dynamics of transposons (TEs) and natural selection. Population studies have found thousands of individual TE insertions in the form of common genetic variants, i.e., TE polymorphisms. Active human TE families include Alu, L1, and SVA elements. These active families of human TE are retrotransposons. Analysis of human polyTE genotypes shows that patterns of TE polymorphism repeat the pattern of human evolution and migration over the past 60,000-100,000 years. They are involved in changes in human regulatory genes. The similarity of patterns allows one to see the effect of TE on regulatory structures that create the structure of the human body, using encoded structures. This conclusion is consistent with studies of intelligence genes, which are based on SNP associations with IQ, as well as with the foundations of a structural and functional network. High proportion of positive selection of genetic variants of our species for the last 6 million years and soft sweeps may explain the accelerated evolution of H. sapiens. The acceleration of gene variability in HAR occurred in parallel with an increase in the activity of the prehuman aimed at the expedient creation of a local environment with neutral mutant genes, expressed in soft sweeps. <i>Humanity itself creates its own present and future biological evolution</i>.

Promoter methylation, transcription, and retrotransposition of LINE-1 in colorectal adenomas and adenocarcinomas

BackgroundThe methylation of the CpG islands of the LINE-1 promoter is a tight control mechanism on the function of mobile elements. However, simultaneous quantification of promoter methylation and transcription of LINE-1 has not been performed in progressive stages of colorectal cancer. In addition, the insertion of mobile elements in the genome of advanced adenoma stage, a precancerous stage before colorectal carcinoma has not been emphasized. In this study, we quantify promoter methylation and transcripts of LINE-1 in three stages of colorectal non-advanced adenoma, advanced adenoma, and adenocarcinoma. In addition, we analyze the insertion of LINE-1, Alu, and SVA elements in the genome of patient tumors with colorectal advanced adenomas.MethodsLINE-1 hypomethylation status was evaluated by absolute quantitative analysis of methylated alleles (AQAMA) assay. To quantify the level of transcripts for LINE-1, quantitative RT-PCR was performed. To find mobile element insertions, the advanced adenoma tissue samples were subjected to whole genome sequencing and MELT analysis.ResultsWe found that the LINE-1 promoter methylation in advanced adenoma and adenocarcinoma was significantly lower than that in non-advanced adenomas. Accordingly, the copy number of LINE-1 transcripts in advanced adenoma was significantly higher than that in non-advanced adenomas, and in adenocarcinomas was significantly higher than that in the advanced adenomas. Whole-genome sequencing analysis of colorectal advanced adenomas revealed that at this stage polymorphic insertions of LINE-1, Alu, and SVA comprise approximately 16%, 51%, and 74% of total insertions, respectively.ConclusionsOur correlative analysis showing a decreased methylation of LINE-1 promoter accompanied by the higher level of LINE-1 transcription, and polymorphic genomic insertions in advanced adenoma, suggests that the early and advanced polyp stages may host very important pathogenic processes concluding to cancer.

Expression dynamics of repetitive DNA in early human embryonic development

BackgroundThe last decade witnessed a number of genome-wide studies on human pre-implantation, which mostly focused on genes and provided only limited information on repeats, excluding the satellites. Considering the fact that repeats constitute a large portion of our genome with reported links to human physiology and disease, a thorough understanding of their spatiotemporal regulation during human embryogenesis will give invaluable clues on chromatin dynamics across time and space. Therefore, we performed a detailed expression analysis of all repetitive DNA elements including the satellites across stages of human pre-implantation and embryonic stem cells.ResultsWe uncovered stage-specific expressions of more than a thousand repeat elements whose expressions fluctuated with a mild global decrease at the blastocyst stage. Most satellites were highly expressed at the 4-cell level and expressions of ACRO1 and D20S16 specifically peaked at this point. Whereas all members of the SVA elements were highly upregulated at 8-cell and morula stages, other transposons and small RNA repeats exhibited a high level of variation among their specific subtypes. Our repeat enrichment analysis in gene promoters coupled with expression correlations highlighted potential links between repeat expressions and nearby genes, emphasising mostly 8-cell and morula specific genes together with SVA_D, LTR5_Hs and LTR70 transposons. The DNA methylation analysis further complemented the understanding on the mechanistic aspects of the repeatome’s regulation per se and revealed critical stages where DNA methylation levels are negatively correlating with repeat expression.ConclusionsTaken together, our study shows that specific expression patterns are not exclusive to genes and long non-coding RNAs but the repeatome also exhibits an intriguingly dynamic pattern at the global scale. Repeats identified in this study; particularly satellites, which were historically associated with heterochromatin, and those with potential links to nearby gene expression provide valuable insights into the understanding of key events in genomic regulation and warrant further research in epigenetics, genomics and developmental biology.

Identification of charged amino acids required for nuclear localization of human L1 ORF1 protein

BackgroundLong Interspersed Element 1 (LINE-1) is a retrotransposon that is present in 500,000 copies in the human genome. Along with Alu and SVA elements, these three retrotransposons account for more than a third of the human genome sequence. These mobile elements are able to copy themselves within the genome via an RNA intermediate, a process that can promote genome instability. LINE-1 encodes two proteins, ORF1p and ORF2p. Association of ORF1p, ORF2p and a full-length L1 mRNA in a ribonucleoprotein (RNP) particle, L1 RNP, is required for L1 retrotransposition. Previous studies have suggested that fusion of a tag to L1 proteins can interfere with L1 retrotransposition.ResultsUsing antibodies detecting untagged human ORF1p, western blot analysis and manipulation of ORF1 sequence and length, we have identified a set of charged amino acids in the C-terminal region of ORF1p that are important in determining its subcellular localization. Mutation of 7 non-identical lysine residues is sufficient to make the resulting ORF1p to be predominantly cytoplasmic, demonstrating intrinsic redundancy of this requirement. These residues are also necessary for ORF1p to retain its association with KPNA2 nuclear pore protein. We demonstrate that this interaction is significantly reduced by RNase treatment. Using co-IP, we have also determined that human ORF1p associates with all members of the KPNA subfamily.ConclusionsThe prediction of NLS sequences suggested that specific sequences within ORF1p could be responsible for its subcellular localization by interacting with nuclear binding proteins. We have found that multiple charged amino acids in the C-terminus of ORF1p are involved in ORF1 subcellular localization and interaction with KPNA2 nuclear pore protein. Our data demonstrate that different amino acids can be mutated to have the same phenotypic effect on ORF1p subcellular localization, demonstrating that the net number of charged residues or protein structure, rather than their specific location, is important for the ORF1p nuclear localization. We also identified that human ORF1p interacts with all members of the KPNA family of proteins and that multiple KPNA family genes are expressed in human cell lines.

Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition.

Long interspersed element 1 (L1) is the only currently active autonomous retroelement in the human genome. Along with the parasitic SVA and short interspersed element Alu, L1 is the source of DNA damage induced by retrotransposition: a copy-and-paste process that has the potential to disrupt gene function and cause human disease. The retrotransposition process is dependent upon the ORF2 protein (ORF2p). However, it is unknown whether most of the protein is important for retrotransposition. In particular, other than the Cys motif, the C terminus of the protein has not been intensely examined in the context of retrotransposition. Using evolutionary analysis and the Alu retrotransposition assay, we sought to identify additional amino acids in the C terminus important for retrotransposition. Here, we demonstrate that Gal4-tagged and untagged C-terminally truncated ORF2p fragments possess residual potential to drive Alu retrotransposition. Using sight-directed mutagenesis we identify that while the Y1180 amino acid is important for ORF2p- and L1-driven Alu retrotransposition, a mutation at this position improves L1 retrotransposition. Even though the mechanism of the contribution of Y1180 to Alu and L1 mobilization remains unknown, experimental evidence rules out its direct involvement in the ability of the ORF2p reverse transcriptase to generate complementary DNA. Additionally, our data support that ORF2p amino acids 1180 and 1250-1262 may be involved in the reported ORF1p-mediated increase in ORF2p-driven Alu retrotransposition.

Potential impact of primate-specific SVA retrotransposons during the evolution of human cognitive function

The SVA family of hominid-specific non-LTR retrotransposon comprises the youngest group of transposable elements in the human genome. The propagation of the most ancient SVA subfamily took place about 13.5 million years ago, and the youngest SVA subfamily appeared in the human genome after the human/chimpanzee divergence. Functional analysis of genes associated with SVA insertions demonstrated their link to multiple ontological categories, with one of the major categories being attributed to brain function. Further analysis of this subset demonstrated that SVA elements expanded their presence in the human genome at different stages of hominoid evolution and were associated with progressively evolving behavioral features that indicate a potential impact of SVA propagation on the cognitive ability of a modern human. Our analysis suggests a potential role of SVAs in the evolution of human central nervous system and especially in the emergence of functional trends relevant to social and parental behavior. Coevolution of behavioral features and reproductive functions are suggested by our analysis and discussed.

The Engineered SVA Trans-mobilization Assay.

Mammalian genomes harbor autonomous retrotransposons coding for the proteins required for their own mobilization, and nonautonomous retrotransposons, such as the human SVA element, which are transcribed but do not have any coding capacity. Mobilization of nonautonomous retrotransposons depends on the recruitment of the protein machinery encoded by autonomous retrotransposons. Here, we summarize the experimental details of SVA trans-mobilization assays which address multiple questions regarding the biology of both nonautonomous SVA elements and autonomous LINE-1 (L1) retrotransposons. The assay evaluates if and to what extent a noncoding SVA element is mobilized in trans by the L1-encoded protein machinery, the structural organization of the resulting marked de novo insertions, if they mimic endogenous SVA insertions and what the roles of individual domains of the nonautonomous retrotransposon for SVA mobilization are. Furthermore, the highly sensitive trans-mobilization assay can be used to verify the presence of otherwise barely detectable endogenously expressed functional L1 proteins via their marked SVA trans-mobilizing activity.

Guanine quadruplexes are formed by specific regions of human transposable elements

BackgroundTransposable elements form a significant proportion of eukaryotic genomes. Recently, Lexa et al. (Nucleic Acids Res 42:968-978, 2014) reported that plant long terminal repeat (LTR) retrotransposons often contain potential quadruplex sequences (PQSs) in their LTRs and experimentally confirmed their ability to adopt four-stranded DNA conformations.ResultsHere, we searched for PQSs in human retrotransposons and found that PQSs are specifically localized in the 3’-UTR of LINE-1 elements, in LTRs of HERV elements and are strongly accumulated in specific regions of SVA elements. Circular dichroism spectroscopy confirmed that most PQSs had adopted monomolecular or bimolecular guanine quadruplex structures. Evolutionarily young SVA elements contained more PQSs than older elements and their propensity to form quadruplex DNA was higher. Full-length L1 elements contained more PQSs than truncated elements; the highest proportion of PQSs was found inside transpositionally active L1 elements (PA2 and HS families).ConclusionsConservation of quadruplexes at specific positions of transposable elements implies their importance in their life cycle. The increasing quadruplex presence in evolutionarily young LINE-1 and SVA families makes these elements important contributors toward present genome-wide quadruplex distribution.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1032) contains supplementary material, which is available to authorized users.

Tangram: a comprehensive toolbox for mobile element insertion detection.

BackgroundMobile elements (MEs) constitute greater than 50% of the human genome as a result of repeated insertion events during human genome evolution. Although most of these elements are now fixed in the population, some MEs, including ALU, L1, SVA and HERV-K elements, are still actively duplicating. Mobile element insertions (MEIs) have been associated with human genetic disorders, including Crohn’s disease, hemophilia, and various types of cancer, motivating the need for accurate MEI detection methods. To comprehensively identify and accurately characterize these variants in whole genome next-generation sequencing (NGS) data, a computationally efficient detection and genotyping method is required. Current computational tools are unable to call MEI polymorphisms with sufficiently high sensitivity and specificity, or call individual genotypes with sufficiently high accuracy.ResultsHere we report Tangram, a computationally efficient MEI detection program that integrates read-pair (RP) and split-read (SR) mapping signals to detect MEI events. By utilizing SR mapping in its primary detection module, a feature unique to this software, Tangram is able to pinpoint MEI breakpoints with single-nucleotide precision. To understand the role of MEI events in disease, it is essential to produce accurate individual genotypes in clinical samples. Tangram is able to determine sample genotypes with very high accuracy. Using simulations and experimental datasets, we demonstrate that Tangram has superior sensitivity, specificity, breakpoint resolution and genotyping accuracy, when compared to other, recently developed MEI detection methods.ConclusionsTangram serves as the primary MEI detection tool in the 1000 Genomes Project, and is implemented as a highly portable, memory-efficient, easy-to-use C++ computer program, built under an open-source development model.

The Minimal Active Human SVA Retrotransposon Requires Only the 5′-Hexamer and Alu-Like Domains

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.

Transcriptional regulation of human-specific SVAF1 retrotransposons by cis-regulatory MAST2 sequences

SVA elements represent the youngest family of hominid non-LTR retrotransposons. Recently, a human-specific subfamily (termed SVAF1, CpG-SVA, or MAST2-SVA) was discovered representing fusion of the CpG island-containing exon 1 of the MAST2 gene and a 5′-truncated SVA. SVAF1 includes at least 84 members, which suggests exceptionally high retrotransposition level. We investigated if the acquirement of the MAST2 CpG-island might play a role in the success of the SVAF1 subfamily. We observed that in 16 samples representing seven human tissues, MAST2 was cotranscribed with the members of the SVAF1 subfamily, but not with other retrotransposons. We found that the methylation status of the MAST2-derived sequences of SVAF1 elements reversely correlates with the transcriptional activity of MAST2. The MAST2 sequence at the 5′ end of SVAF1 acts as a positive transcriptional regulator in human germ cells. Finally, in various testicular tissue samples we uncovered a transcriptional correlation of MAST2 with the human L1, Alu and SVA retrotransposons.