Variation in Base Composition Underlies Functional and Evolutionary Divergence in Non-LTR Retrotransposons

ABSTRACTBackgroundNon-LTR retrotransposons often exhibit base composition that is markedly different from the nucleotide content of their host’s gene. For instance, the mammalian L1 element is AT-rich with a strong A bias on the positive strand, which results in a reduced transcription. It is plausible that the A-richness of mammalian L1 is a self-regulatory mechanism reflecting a trade-off between transposition efficiency and the deleterious effect of L1 on its host. We examined if the A-richness of L1 is a general feature of non-LTR retrotransposons or if different clades of elements have evolved different nucleotide content. We also investigated if elements belonging to the same clade evolved towards different base composition in different genomes or if elements from the same clades evolved towards similar base composition in the same genome.ResultsWe found that non-LTR retrotransposons differ in base composition among clades within the same host but also that elements belonging to the same clade differ in base composition among hosts. We showed that nucleotide content remains constant within the same host over extended period of evolutionary time, despite mutational patterns that should drive nucleotide content away from the observed base composition.ConclusionsOur results suggest that base composition is evolving under selection and may be reflective of the long-term co-evolution between non-LTR retrotransposons and their host. Finally, the coexistence of elements with drastically different base composition suggests that these elements may be using different strategies to persist and multiply in the genome of their host.

Formation of the L1Hs retroelement in the intron of the MGMT gene of hominoidea

Aim. Analyze the formation of a human-specific L1Hs element in the intron 3 of the MGMT gene on an example of a hominid. Methods. The results of the search and identification of mobile genetic elements were performed using the CENSOR program. The homology between nucleotide sequences was determined by BLAST 2.6.1. Results. The components of the cluster, where the L1Hs element in the human being was formed, are fragments of the L1PA6 element, which are common in the monkeys of the Old and New World. In the gibbon, among the L1 element groups, there are representatives of older subfamilies (L1PB, L1MC, L1MD and L1ME), and the partial homology to the L1Hs of the element is predominantly of elements of groups that have arisen in the mammalian genomes. Conclusions. Formation of a human-specific L1Hs element occurred during the evolution of Hominoidea in parallel with the formation of the cluster structure of MGE in humans from different subfamilies of LINE1-elements whose component components, obviously, also involved in the formation of the L1Hs element. Keywords: Hominoidea, MGMT gene, intron 3, human-specific L1Hs element.

2057 L1 expression analysis in adipose-derived stem cells

OBJECTIVES/SPECIFIC AIMS: Long interspersed element-1s (L1s) are autonomous, mobile elements that are able to copy and insert themselves throughout the genome with their own reverse transcriptase and endonuclease. These elements make up 17% of the human genome with over 500,000 copies, though the vast majority of L1s are defective with only a few dozen potentially responsible for L1 activity. Full-length L1s have the potential to contribute to mutagenesis through random insertion and increased genetic instability. Here we set out to study L1 expression at the specific loci level in bone marrow-derived stem cells (bmSCs) and adipose-derived stem cells (ASCs) and compare the levels of expression from ASCs from donor patients who are young and lean, obese, and old. Our hypothesis is that L1-related damage may contribute to mutation and inflammation that alters the function of these stem cells throughout the life of an individual. METHODS/STUDY POPULATION: ASCs and bmSCs were isolated from patient donors. The following samples were collected: ASCs from 3 young (under the age of 59) and lean (BMI<30) patients, ASCs from 3 older patients (over the age of 59), ASCs from 3 patients with BMI>30, and bmSCs from 4 young and lean patients. Cytoplasmic RNA from the cell populations was isolated and sequenced by RNA-Seq from the cell populations. Using our recently developed bioinformatics pipeline, we set out to quantify L1 expression and identify the few culprit L1s at specific loci that are actively transcribing to RNA in the ASC and bmSC samples. RESULTS/ANTICIPATED RESULTS: Here we provide proof of concept with the application of this novel method in characterizing full-length expressed L1s at the specific loci level in ASCs and bmSCs. We identified L1 loci that are commonly expressed in these cell types and observed an increase in L1 expression in the obese and old ASC cells compared with the young, lean ASCs and bmSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: ASCs hold the promise of broad application in the biomedical field including regenerative treatment. There are reports that ASCs cultivated from older and obese donors are less effective in regenerative treatments. By demonstrating an increased expression of the mutagenic L1 element in ASCs from obese and old donors, this study provides further evidence suggesting the preferable use of ASCs from young and lean donors for regenerative therapies. These studies will also help us to understand the potential contribution of L1 expression to loss of stem cell function during the aging process.

MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by downregulating the nuclear import factor TNPO1

ABSTRACTRepetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements are repressed by distinct molecular mechanisms including DNA methylation and histone modifications to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells) a window of opportunity for L1 de-repression arises and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3’UTR of the nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-RNP complex (using L1-encoded ORF1p as a proxy for L1-RNP complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA, and regulating a cellular factor necessary for L1 nuclear import and retrotransposition.

L1 Retrotransposons Are Transcriptionally Active in Hippocampus of Rat Brain

LINE1 (L1) is an autonomous, non-LTR retrotransposon and the L1 family of retrotransposons constitute around 17%, 20% and 23% in the human, mouse and rat genomes respectively. Under normal physiological conditions, the retroelements remain by and large transcriptionally silent but are activated in response to biotic and abiotic stress conditions and during perturbation in cellular metabolism. They have also been shown to be transiently activated under certain developmental programs. Using RT-PCR, we show that the L1 elements are transcriptionally active in the hippocampus region of the brain of four-month-old rat under normal conditions without any apparent stress. Twenty non-redundant LINE1-specific reverse transcriptase (RTase) sequences form ORF2 region were isolated, cloned and sequenced. Full length L1 element sequences complementary to the isolated sequences were retrieved from the L1 database. In silico analysis was used to determine the presence of these retroelements proximal (up to 10 kb) to the genes transcriptionally active in the hippocampus. Many important genes were found to be in close proximity of the transcriptionally active L1 elements. Transcriptional activation of the elements possibly affects the expression of the neighbouring genes.

Identification and Characterization of L1-Mediated Large Tandem Duplication, Spanning Exon 4 to 10 of the F13A1, In a Patient with Congenital Factor XIII Deficiency.

Abstract 3659 Blood coagulation factor XIII (FXIII) is a plasma glycoprotein that plays an important role in the stabilization of fibrin clot in the final stage of blood coagulation. The FXIII circulates as a heterotetramer composed of two A and two B subunits in plasma. The A subunit (FXIIIA) possesses catalytic activity and this catalytic subunit is carried and protected by the B subunit (FXIIIB). Inherited deficiency of FXIII is a rare autosomal recessive bleeding disorder. Based on the genotype, it is classified into two types: FXIIIA deficiency (>95% of all cases), characterized by mutations in the F13A1 gene, and FXIIIB deficiency, characterized by mutations in the F13B gene. At the time of writing, at least 86 different mutations, most of which are point mutations, have been identified and registered in the F13-database. Here we show a novel, large tandem duplication in the F13A1 gene of a patient with congenital factor XIII deficiency. A female patient, born to consanguineous parents, suffered from severe bleeding diathesis, including menorrhagia, intracranial hemorrhage and ovarian hemorrhage, from childhood. Bleeding manifestations had been successfully controlled by monthly prophylactic replacement therapy using factor XIII concentrate (Fibrogammin). Trough levels of both factor XIII activity and antigen were 19% (Berichrom FXIII) and <10% (ELISA method), respectively. No pathogenic mutations associated with FXIII deficiency were detected from nucleotide sequencing of the coding region, 5′-UTR and 3′-UTR of both F13A1 and F13B. However, from an observation of the RT-PCR amplification state, the F13A1 mRNA level of the patient was apparently lower than that of healthy individuals. This result suggested the existence of abnormalities in the patient's F13A1. Relative exon copy number analysis using real-time PCR revealed two times as many of the continuing 7 exons (exon 4–10) as in the remaining region of the F13A1. This is likely due to some genomic rearrangement, probably homologous recombination in the F13A1 gene. IVS-3 and IVS-10 of F13A1 were very large and contained many repetitive sequences. The existence of an almost full-length (≂f6kb) L1 element, a well known long interspersed repetitive element (LINE) in humans, in both introns suggests that the recombination might arise from the L1 element. The provision of the PCR product (amplified by an IVS-10 specific forward primer and an IVS-3 specific reverse primer) confirmed that IVS-10 connected to IVS-3 with L1 as the boundary. Furthermore, sequencing this PCR product identified the 15bp sequence in the L1 element as an actual breakpoint. Taken together with the results of the analysis of the genomic DNA, this confirmed that the L1-mediated large (≂f109kb) tandem duplication was located in the patient's F13A1. In order to quantify the F13A1 mRNA, a relative real-time PCR quantification was performed using TaqMan Gene Expression Assays. Three different positions: one upstream of duplication (Exon2-3), one at duplication (Exon5-6) and one downstream of the duplication (Exon13-14) were used. The mRNA level of the patient was a markedly low compared to the normal control and the duplicated region (28% of normal) was clearly higher than both the upstream and downstream positions (9% of normal for each). This result reflected that the mRNA was probably maintaining the duplication. In order to analyze the mRNA splicing of the joint between the two duplicated regions, RT-PCR using a forward primer in exon 9 and a reverse primer in exon 4 of F13A1 was performed. Two major transcripts were amplified. The larger transcript was the product that keeps the genomic exon order and connects exon 10 to 4. However, the transcript is thought to lead to frameshift and to generate premature termination codon in exon 4. The other was the product that was skipping exon 10 and connected exon 9 to 4. This transcript is thought to escape frameshift and may translate to the unusual extra large FXIIIA. However, it is unlikely that the protein translated from the extra large mRNA circulates in blood. In conclusion, we identified an L1-mediated large tandem duplication, spanning exon 4 to 10 of the F13A1 gene, as an etiology of the congenital FXIII deficiency. Disclosures: No relevant conflicts of interest to declare.