Long INterspersed Element-1 (L1) retrotransposons use activities contained within the L1 open reading frame 2-encoded protein (ORF2p) to mobilize throughout the genome via target-site primed reverse transcription (TPRT). The ORF2p endonuclease domain (EN) cleaves genomic DNA to liberate a 3'-hydroxyl (3'-OH) group that is used by the ORF2p reverse transcriptase domain (RT) to synthesize a cDNA copy of its bound L1 RNA template. L1 also can move by EN-independent retrotransposition (ENi), where a 3'-OH group at genomic DNA lesions, dysfunctional telomeres, or stalled replication forks is proposed to prime L1 reverse transcription in the absence of L1 EN cleavage. We previously reported that ribonucleoprotein (RNP) preparations from cells transfected with a human wild-type (WT) L1 or L1 EN-mutant, but not an L1 RT-mutant, can initiate reverse transcription from a DNA oligonucleotide primer/L1 RNA template complex. The WT and EN-deficient L1 RNP preparations also are associated with a nuclease activity that can process a 3' end modification that precludes DNA synthesis from an oligonucleotide prior to priming the L1 RT reaction. Here, we purified recombinant full-length WT, L1 EN-, and L1 RT-mutant human L1 ORF2p from insect cells. We report that the WT and L1 EN-mutant, but not the L1 RT-mutant, contain an alternative endonuclease activity (alt-EN). Alt-EN activity also is detected in a bacterially expressed L1 ORF2p protein that lacks the L1 EN and ORF2p cysteine-rich domains and a thermostable group II intron-encoded protein. Processing of diverse modified primers demonstrates endonucleolytic cleavage that is eliminated by mutations in the RT active site. We propose that alt-EN is an evolutionarily conserved activity within the RT fold that promoted ENi retrotransposition of primordial retrotransposons prior to the acquisition of an EN domain.

Due to their history of domestication and breed formation, dogs are a powerful system for studying the phenotypic impact of genetic variation. Comparison of canine genome assemblies show that retrotransposons, mobile elements that mobilize via an RNA intermediate, are a major contributor to canine genetic diversity with an eightfold and 17-fold increase of LINE-1 and SINE differences, respectively, in dogs relative to that found among humans. The frequency of dimorphic retrotransposon insertions among dogs suggests these elements have mobilized at a high rate over recent canine evolution. However, the rate at which new insertions arise has yet to be determined. We aligned a collection of genome assemblies derived from four breed dogs, a Dingo, and an American grey wolf to a Greenland grey wolf to identify dimorphic LINE-1 and SINEC insertions. Across our panel of seven canine assemblies, we identified and characterized 7,428 dimorphic LINE-1s and 51,572 dimorphic SINECs. Each assembly differs from the Greenland wolf genome by an average of 3,497 LINE-1s and 25,558 SINECs. Analysis of allele sharing among samples recapitulates known relationships and reveals substantial within-breed variation. Calibrating estimates using a previously estimated single nucleotide mutation rate of 4.5 × 10− 9 per base pair per generation, we estimate that new LINE-1 and SINEC and insertions have occurred at a rate of 1/184 and 1/22 births over recent canine evolution. These estimates are largely consistent across assemblies and breeds. Our phylogenetic estimate of SINEC retrotransposition in canines is approximately twice as large as that estimated for Alu elements in humans, while the canine LINE-1 insertion rate is within the range of human estimates. These data suggest that although SINEC has been an outsized driver of canine genome evolution, the striking levels of canine LINE-1 and SINEC dimorphism mainly reflect high levels of long-standing genetic variation.

X-chromosome inactivation (XCI) enables gene dosage compensation in XX eutherians. Long interspersed element-1 (LINE-1 or L1) retrotransposons are unusually abundant on the human X chromosome and are hypothesized to facilitate XCI. Here, we used long-read DNA sequencing to conduct a haplotype-aware analysis of engineered L1 integration preferences in the PA-1 human embryonic carcinoma cell line. Crucially, clonal XCI in PA-1 cells enabled derivation of active (Xa) and inactive (Xi) X-chromosome haplotypes. L1 integration strongly favored the Xi and other genomic regions that undergo DNA replication late in S-phase. These results suggest that the X chromosome is L1 rich because of XCI and imply that L1 integration preference for the Xi in XX individuals could potentially double the frequency of X-linked pathogenic L1 mutations in their XY descendants.

Alu elements are primate-specific retrotransposons that comprise ∼11% of human DNA. Alu sequences contain an internal RNA polymerase III promoter, and the resultant Alu RNA transcripts mobilize by a replicative process termed retrotransposition, which requires the long interspersed element-1 open reading frame 2-encoded protein (ORF2p). Here, we used HeLa cell-based retrotransposition assays to define a minimal Alu domain necessary for retrotransposition. We demonstrate that Alu transcripts expressed from a cytomegalovirus (CMV) RNA polymerase II promoter can efficiently undergo retrotransposition. The use of an external CMV promoter to express Alu RNA allowed us to construct separation-of-function mutations to examine the effects of large deletions within the Alu sequence on retrotransposition. Deletion mutagenesis demonstrated that a 46-nucleotide (nt) domain located at the 5′ end of the Alu RNA transcript is necessary for retrotransposition. Consistent with current models, the 46-nt 5′ Alu domain associates with SRP9/14 in HeLa-HA cell extracts and can promote retrotransposition in HeLa-HA cells. We propose that the 46-nt 5′ Alu domain forms a discrete structure that allows for SRP9/14 binding and ribosomal association, thereby allowing the Alu poly(A) tract to compete with the L1 poly(A) tail for ORF2p RNA binding to mediate its retrotransposition.

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1 or L1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing messenger RNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINE RNAs use strategies to hijack L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.

When somatic cells acquire complex karyotypes, they often are removed by the immune system. Mutant somatic cells that evade immune surveillance can lead to cancer. Neurons with complex karyotypes arise during neurotypical brain development, but neurons are almost never the origin of brain cancers. Instead, somatic mutations in neurons can bring about neurodevelopmental disorders, and contribute to the polygenic landscape of neuropsychiatric and neurodegenerative disease. A subset of human neurons harbors idiosyncratic copy number variants (CNVs, "CNV neurons"), but previous analyses of CNV neurons are limited by relatively small sample sizes. Here, we develop an allele-based validation approach, SCOVAL, to corroborate or reject read-depth based CNV calls in single human neurons. We apply this approach to 2,125 frontal cortical neurons from a neurotypical human brain. SCOVAL identifies 226 CNV neurons, which include a subclass of 65 CNV neurons with highly aberrant karyotypes containing whole or substantial losses on multiple chromosomes. Moreover, we find that CNV location appears to be nonrandom. Recurrent regions of neuronal genome rearrangement contain fewer, but longer, genes.

Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Retrotransposons can cause somatic genome variation in the human nervous system, which is hypothesized to have relevance to brain development and neuropsychiatric disease. However, the detection of individual somatic mobile element insertions presents a difficult signal-to-noise problem. Using a machine-learning method (RetroSom) and deep whole-genome sequencing, we analyzed L1 and Alu retrotransposition in sorted neurons and glia from human brains. We characterized two brain-specific L1 insertions in neurons and glia from a donor with schizophrenia. There was anatomical distribution of the L1 insertions in neurons and glia across both hemispheres, indicating retrotransposition occurred during early embryogenesis. Both insertions were within the introns of genes ( CNNM2 and FRMD4A ) inside genomic loci associated with neuropsychiatric disorders. Proof-of-principle experiments revealed these L1 insertions significantly reduced gene expression. These results demonstrate that RetroSom has broad applications for studies of brain development and may provide insight into the possible pathological effects of somatic retrotransposition.

Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we use immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2′−5′-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5′UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s lead to a modest induction of IFN-α expression, which is abrogated upon HELZ2 overexpression. Notably, IFN-α expression is enhanced upon overexpression of an ORF1p RNA binding mutant, suggesting ORF1p binding might protect L1 RNA from “triggering” IFN-α induction. Thus, ISG proteins can inhibit retrotransposition by different mechanisms.

When somatic cells acquire complex karyotypes, they are removed by the immune system. Mutant somatic cells that evade immune surveillance can lead to cancer. Neurons with complex karyotypes arise during neurotypical brain development, but neurons are almost never the origin of brain cancers. Instead, somatic mutations in neurons can bring about neurodevelopmental disorders, and contribute to the polygenic landscape of neuropsychiatric and neurodegenerative disease. A subset of human neurons harbors idiosyncratic copy number variants (CNVs, "CNV neurons"), but previous analyses of CNV neurons have been limited by relatively small sample sizes. Here, we developed an allele-based validation approach, SCOVAL, to corroborate or reject read-depth based CNV calls in single human neurons. We applied this approach to 2,125 frontal cortical neurons from a neurotypical human brain. This approach identified 226 CNV neurons, as well as a class of CNV neurons with complex karyotypes containing whole or substantial losses on multiple chromosomes. Moreover, we found that CNV location appears to be nonrandom. Recurrent regions of neuronal genome rearrangement contained fewer, but longer, genes.

Abstract Copy number variation (CNV) may lead to pathological traits, and Charcot-Marie-Tooth disease type 1A (CMT1A), the commonest inherited peripheral neuropathy, is due to a genomic duplication encompassing the dosage-sensitive PMP22 gene. MicroRNAs act as repressors on post-transcriptional regulation of gene expression and in rodent models of CMT1A, overexpression of one such microRNA (miR-29a) has been shown to reduce the PMP22 transcript and protein level. Here we present genomic and functional evidence, for the first time in a human CNV-associated phenotype, of the 3′ untranslated region (3′-UTR)-mediated role of microRNA repression on gene expression. The proband of the family presented with an early-onset, severe sensorimotor demyelinating neuropathy and harboured a novel de novo deletion in the PMP22 3′-UTR. The deletion is predicted to include the miR-29a seed binding site and transcript analysis of dermal myelinated nerve fibres using a novel platform, revealed a marked increase in PMP22 transcript levels. Functional evidence from Schwann cell lines harbouring the wild-type and mutant 3′-UTR showed significantly increased reporter assay activity in the latter, which was not ameliorated by overexpression of a miR-29a mimic. This shows the importance of miR-29a in regulating PMP22 expression and opens an avenue for therapeutic drug development.

Cells elaborate transcriptional programs in response to external signals. In the peripheral nerves, Schwann cells (SC) sort axons of given caliber and start the process of wrapping their membrane around them. We identify Actin-like protein 6a (ACTL6a), part of SWI/SNF chromatin remodeling complex, as critical for the integration of axonal caliber recognition with the transcriptional program of myelination. Nuclear levels of ACTL6A in SC are increased by contact with large caliber axons or nanofibers, and result in the eviction of repressive histone marks to facilitate myelination. Without Actl6a the SC are unable to coordinate caliber recognition and myelin production. Peripheral nerves in knockout mice display defective radial sorting, hypo-myelination of large caliber axons, and redundant myelin around small caliber axons, resulting in a clinical motor phenotype. Overall, this suggests that ACTL6A is a key component of the machinery integrating external signals for proper myelination of the peripheral nerve.

Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3'UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3'UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3'UTR, and represses L1 retrotransposition. We provide evidence that the condensin I protein, NCAPD2, is required for condensin II and GAIT protein association with L1 RNA. Furthermore, condensin I and condensin II subunits interact to form a L1-dependent super condensin complex (SCC) which is located primarily within the cytoplasm of both transformed and primary epithelial cells. These data suggest that increases in L1 expression in epithelial cells promote cytoplasmic condensin protein associations that facilitate a feedback loop in which condensins may cooperate to mediate L1 repression.

Retrotransposons are selfish genetic elements that encode an enzyme, reverse transcriptase (RT), which converts the element-encoded RNA into DNA prior to or during genomic integration. New studies provide compelling evidence that a bacterial group II intron-like RT has adapted enzymatic activities associated with RTs to function in host DNA repair.