Biology subjects

Moran, J. V.

Publications and source records attributed to Moran, J. V..

7 recordsLinked to original sources

bioRxiv2026

An Alternative DNA Endonuclease Activity is Associated with the LINE-1 ORF2-encoded Protein

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.

molecular biology↗

bioRxiv2024

Identification of a minimal Alu domain required for retrotransposition

Alu elements are primate-specific retrotransposon sequences that comprise [~]11% of human genomic DNA. Alu sequences contain an internal RNA polymerase III promoter and the resultant Alu RNA transcripts mobilize by a replicative process termed retrotransposition. Alu retrotransposition requires the Long INterspersed Element-1 (LINE-1) open reading frame 2-encoded protein (ORF2p). Current models propose that Alu RNA binds to signal recognition particle proteins 9 and 14 (SRP9/14) and localizes to ribosomes, which allows Alu to hijack L1 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 Alu retrotransposition. Consistent with current models, the 46 nt 5' Alu domain associates with SRP9/14 in HeLa-HA cell extracts and can promote a single round of retrotransposition in HeLa-HA cells. We propose that the 46 nt 5' Alu domain forms a discrete structure that allows for SRP 9/14 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.

molecular biology↗

bioRxiv2023

Mapping the Complex Genetic Landscape of Human Neurons

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.

neuroscience↗

bioRxiv2022

The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction

Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we used 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 5UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s led to a modest induction of IFN- expression, which was abrogated upon HELZ2 overexpression. Notably, IFN- expression was enhanced upon overexpression of an ORF1p RNA binding mutant, suggesting ORF1p binding might protect L1 RNA from "triggering" IFN- induction. Thus, ISG proteins can inhibit retrotransposition by different mechanisms.

molecular biology↗

bioRxiv2020

Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long non-coding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3 end of LINE-1_Cfs (i.e., LINE-1_Cf 3-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation. SignificanceAdvancements in long-read DNA sequencing technologies provide more comprehensive views of genomes. We used long-read sequences to assemble a Great Dane dog genome that provides several improvements over the existing reference derived from a Boxer dog. Assembly comparisons revealed that gaps in the Boxer assembly often occur at the beginning of protein-coding genes and have a high-GC content, which likely reflects limitations of previous technologies in resolving GC-rich sequences. Dimorphic LINE-1 and SINEC retrotransposon sequences represent the predominant differences between the Great Dane and Boxer assemblies. Proof-of-principle experiments demonstrated that expression of a canine LINE-1 could promote the retrotransposition of itself and a SINEC_Cf consensus sequence in cultured human cells. Thus, ongoing retrotransposon activity may contribute to canine genetic diversity.

genomics↗

bioRxiv2019

Machine learning reveals bilateral distribution of somatic L1 insertions in human neurons and glia

Active retrotransposons in the human genome (L1, Alu and SVA elements) can create genomic mobile element insertions (MEIs) in both germline and somatic tissue1. Specific somatic MEIs have been detected at high levels in human cancers2, and at lower to medium levels in human brains3. Dysregulation of somatic retrotransposition in the human brain has been hypothesized to contribute to neuropsychiatric diseases4, 5. However, individual somatic MEIs are present in small proportions of cells at a given anatomical location, and thus standard whole-genome sequencing (WGS) presents a difficult signal-to-noise problem, while single-cell approaches suffer from limited scalability and experimental artifacts introduced by enzymatic whole-genome amplification6. Previous studies produced widely differing estimates for the somatic retrotransposition rates in human brain3, 6-8. Here, we present a highly precise machine learning method (RetroSom) to directly identify somatic L1 and Alu insertions in <1% cells from 200x deep WGS, which allows circumventing the restrictions of whole-genome amplification. Using RetroSom we confirmed a lower rate of retrotransposition for individual somatic L1 insertions in human neurons. We discovered that anatomical distribution of somatic L1 insertion is as widespread in glia as in neurons, and across both hemispheres of the brain, indicating retrotransposition occurs during early embryogenesis. We characterized two of the detected brain-specific L1 insertions in great detail in neurons and glia from a donor with schizophrenia. Both insertions are within introns of genes active in brain (CNNM2, FRMD4A) in regions with multiple genetic associations with neuropsychiatric disorders9-11. Gene expression was significantly reduced by both somatic insertions in a reporter assay. Our results provide novel insights into the potential for pathological effects of somatic retrotransposition in the human brain, now including the large glial fraction. RetroSom has broad applicability in all disease states where somatic retrotransposition is expected to play a role, such as autoimmune disorders and cancer.

bioinformatics↗

bioRxiv2018

Satellite DNA-containing gigantic introns in a unique gene expression program during Drosophila spermatogenesis

Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. Here we identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. We find that two Drosophila genes with intron gigantism, kl-3 and kl-5, are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. We identify two RNA-binding proteins that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. We propose that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation.

developmental biology↗