Heritable L1 retrotransposition in the mouse primordial germline and early embryo

LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo

Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle

<p>Abstract</p> <p>Background</p> <p>Mutations in methylmalonyl-CoA mutase cause methylmalonic acidemia, a common organic aciduria. Current treatment regimens rely on dietary management and, in severely affected patients, liver or combined liver-kidney transplantation. For undetermined reasons, transplantation does not correct the biochemical phenotype.</p> <p>Methods</p> <p>To study the metabolic disturbances seen in this disorder, we have created a murine model with a null allele at the methylmalonyl-CoA mutase locus and correlated the results observed in the knock-out mice to patient data. To gain insight into the origin and magnitude of methylmalonic acid (MMA) production in humans with methylmalonyl-CoA mutase deficiency, we evaluated two methylmalonic acidemia patients who had received different variants of combined liver-kidney transplants, one with a complete liver replacement-kidney transplant and the other with an auxiliary liver graft-kidney transplant, and compared their metabolite production to four untransplanted patients with intact renal function.</p> <p>Results</p> <p>Enzymatic, Western and Northern analyses demonstrated that the targeted allele was null and correctable by lentiviral complementation. Metabolite studies defined the magnitude and tempo of plasma MMA concentrations in the mice. Before a fatal metabolic crisis developed in the first 24–48 hours, the methylmalonic acid content per gram wet-weight was massively elevated in the skeletal muscle as well as the kidneys, liver and brain. Near the end of life, extreme elevations in tissue MMA were present primarily in the liver. The transplant patients studied when well and on dietary therapy, displayed massive elevations of MMA in the plasma and urine, comparable to the levels seen in the untransplanted patients with similar enzymatic phenotypes and dietary regimens.</p> <p>Conclusion</p> <p>The combined observations from the murine metabolite studies and patient investigations indicate that during homeostasis, a large portion of circulating MMA has an extra-heptorenal origin and likely derives from the skeletal muscle. Our studies suggest that modulating skeletal muscle metabolism may represent a strategy to increase metabolic capacity in methylmalonic acidemia as well as other organic acidurias. This mouse model will be useful for further investigations exploring disease mechanisms and therapeutic interventions in methylmalonic acidemia, a devastating disorder of intermediary metabolism.</p

Usefulness of a CACA Repeat Polymorphism in Genotype Assignments in Duchenne/Becker Muscular Dystrophy

RFLP analysis in Duchenne/Becker muscular dystrophy (D/BMD) has been limited by the lack of informative marker loci at the 3′ end of the dystrophin gene. Recently a CACA repeat polymorphism was described in the 3′ untranslated end of the dystrophin gene which we have found helpful in genotype assignments of D/BMD families when an RFLP approach is required. The CACA repeat marker has 2 common alleles (1 and 2) that are easily visualized by a nonradioactive PCR method followed by polyacrylamide gel electrophoresis. We present 2 families which demonstrate the use of this polymorphism. Since 35–50% of females are heterozygous, this locus is a useful marker in RFLP analysis of D/BMD families

MOV10 RNA Helicase Is a Potent Inhibitor of Retrotransposition in Cells

<div><p>MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.</p> </div

Construct pc-L1-1FH successfully immunoprecipitates basal L1 RNP complexes (ORF1p, ORF2p, and L1 RNA) from 293T cell lysates following α-FLAG purification.

<p>Detection in purified immunoprecipitates of (A) FLAG-HA-tagged ORF1 protein, (B) ORF2 protein, (C) L1 RNA detected by RT-PCR, and (D) ORF2 reverse transcriptase activity, assayed as described by Kulpa et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002941#pgen.1002941-Kulpa1" target="_blank">[35]</a>. (E–I) Endogenous and ectopically expressed MOV10 protein and L1 ORF1p associate in multiple cell lines. (E) Immunoprecipitation of V5-tagged MOV10 by FLAG-tagged pc-L1-1FH depends upon the presence of RNA (lanes 2 and 3). A double point mutation in ORF1 of pc-L1-1FH known to inhibit RNA-binding, prevents efficient co-IP of MOV10 protein (lane 4). Removing the FLAG-HA-tag from pc-L1-FH (pc-L1-RP) prevents IP of the L1 RNP and MOV10 protein on α-FLAG agarose (lane 5). (F) pc-L1-1FH-generated RNPs associate with endogenous MOV10 protein in 293T cells. (G) Transfected V5-tagged MOV10 and cold shock domain protein YBX1, but not fibrillarin (FBL) or empty vector, immunoprecipitate endogenous ORF1p from 2102Ep cells. (H) α-ORF1 (AH40.1) antibody co-IPs endogenous MOV10 protein from 2102Ep cells (lane 1). Lane 3: lysate of 293T cells transfected with MOV10-V5-His₆ WT as a marker for MOV10 protein. (I) Similarly, immunoprecipitation using α-MOV10 antibody yields endogenous ORF1p (lane 1). Lane 4: α-FLAG-tag IP from 293T cells transfected with pc-L1-1FH as a marker for ORF1p.</p

Polycomb group (PcG) multiprotein PRC1-like complex component Chromobox homolog 7 (CBX7) associates with the L1-RNP and inhibits retrotransposition.

<p>(A) Lanes 1–3: CBX7 binds weakly with the L1 RNP in an RNA-dependent manner. Lanes 4–9: Co-immunoprecipitation of V5-tagged CBX7 by pc-L1-1FH is greatly enhanced by coexpression of tagged MOV10 proteins. (B) When overexpressed in the cell culture assay, both CBX7 and PRC1 component CBX8 significantly inhibit L1 retrotransposition, without obvious cell toxicity (C).</p