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PubMed2002

Generation of two homologous and intronless zinc-finger protein genes, zfp352 and zfp353, with different expression patterns by retrotransposition.

We have previously reported a mouse zinc-finger protein gene, Zfp352 (formerly 2czf48), that is expressed in early mouse embryos. Here, we report the genomic structure of Zfp352 and its lung-specific homolog, Zfp353. The two genes map on different chromosomes at 4C6 and 8B3.1. Both genes are intronless, except for the presence of a single 4.6-kb intron in the 5' untranslated region of Zfp352. The genes use different RNA start sites located 1.2 kb apart within the 5' homologous region. LINE1 sequences are structurally associated with the genes and form an integral part of Zfp353 transcripts, suggesting previous retrotransposition events. We propose a model of evolution of the genes. The main feature of the model is the presence of a fortuitous upstream promoter and an intron in the first retrotransposition site, creating a pre-Zfp352 gene with a 5' untranslated region intron. A second retrotransposition event copying from the pre-Zfp352 retroposon and removing the fortuitous intron resulted in the intronless Zfp353 at a different chromosomal location and with a different mode of expression. The model may be applicable to other genes with a similar structure with a single intron in the 5' untranslated region. The exact role of LINE1 in the retrotransposition events remains to be elucidated.

Animals↗
PubMed2002

Discovery of a functional retrotransposon of the murine phospholipid hydroperoxide glutathione peroxidase: chromosomal localization and tissue-specific expression pattern.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenoprotein capable of reducing toxic hydroperoxy ester lipids, has been implicated in antioxidative defense and spermatogenesis. Screening a murine genomic library, we isolated two recombinants (pseudogenes 1 and 2) containing retrotransposons for this enzyme. On comparison with the paralogous cDNA, pseudogene 1 contained only two silent nucleotide exchanges, and the 3'-untranslated region (3'-UTR) carrying the functionally important selenocysteine insertion sequence was free of mutations. This retrotransposon was found in various mouse strains and could be mapped to the region B2-B3 of chromosome 10. In vitro studies indicated significant promoter activity in the 5'-flanking region of pseudogene 1, and we observed a tissuespecific expression of this retrotransposon. In the submandibular gland. Most PHGPx transcripts originated from pseudogene 1. In contrast, pseudogene 2, containing numerous mutations in all parts of the retrotransposon, was not expressed in any tissue. It was mapped to region E3-E4 of chromosome 17, and we did not detect any promoter activity in its 5'-flanking region. These data indicate the existence of two retrotransposed PHGPx pseudogenes, one of which encodes a functional enzyme. This retrotransposon belongs to the rare group of pseudogenes that are tissue-specifically expressed under the control of captured regulatory elements, and it constitutes an example of evolutionarily acquired redundancy in gene expression. The results are important for the design of future knockout strategies aimed at investigating the biological role of this enzyme.

5' Flanking Region↗
PubMed2002

Many human genes are transcribed from the antisense promoter of L1 retrotransposon.

Human L1 retrotransposon has two transcription-regulatory regions: an internal or sense promoter driving transcription of the full-length L1, and an antisense promoter (ASP) driving transcription in the opposite direction into adjacent cellular sequences yielding chimeric transcripts. Both promoters are located in the 5'-untranslated region (5'-UTR) of L1. Chimeric transcripts derived from the L1 ASP are highly represented in expressed-sequence tag (EST) databases. Using a bioinformatics approach, we have characterized 10 chimeric ESTs (cESTs) derived from the EST division of GenBank. These cESTs contained 3' regions similar or identical to known cellular mRNA sequences. They were accurately spliced and preferentially expressed in tumor cell lines. Analysis of the hundreds of cESTs suggests that the L1 ASP-driven transcription is a common phenomenon not only for tumor cells but also for normal ones and may involve transcriptional interference or epigenetic control of different cellular genes.

5' Flanking Region↗
PubMed2002

A new family of chimeric retrotranscripts formed by a full copy of U6 small nuclear RNA fused to the 3' terminus of l1.

Long interspersed nuclear elements (LINE-1, L1) constitute a large family of mammalian retrotransposons that have been replicating and evolving in mammals for more than 100 million years and now compose 17% of the human genome. They have an important creative role in human genomic evolution through mechanisms such as new integrations, generation of processed pseudogenes, and transfer of non-L1 DNA flanking their 3' ends to new genomic locations. Here we present evidence that the L1 integration machinery was used for the creation of a new family of chimeric retrotranscripts, which contain a full copy of U6 small nuclear RNA and a 3' part of L1 at their 5' and 3' ends, respectively. There are at least 56 members of this family in the human genome. The integrations of such fused retrotranscripts into the human genome took place until recently. Here we report one U6-L1 insertion that is polymorphic in humans. We also propose a mechanism used to generate chimeric retrotranscripts.

Humans↗
PubMed1995

Characterization of a non-long terminal repeat retrotransposon cDNA (L1Tc) from Trypanosoma cruzi: homology of the first ORF with the ape family of DNA repair enzymes.

In the present paper we describe the characterization of a Trypanosoma cruzi cDNA (L1Tc) corresponding to a transcript from a new long terminal repeat (LTR) retrotransposon. This element is present in a high-copy number, and is found dispersed throughout the T. cruzi genome. Northern analysis shows an abundant expression of L1Tc-related sequences with a major band of about 5 kb. The transcript has at its 3' end a fragment of a highly repetitive DNA sequence (E12A), at its 5' end a ribosomal mobile element-like sequence and three putative open reading frames (ORF) in different frames. The ORF2 codes for a protein which has significant homology with the retrotranscriptase-related sequences from non-LTR retrotransposons containing the seven domains present in all the retrotranscriptase and retrotranscriptase-related proteins. The ORF3 codes for a gag-like protein showing unusual cysteine motifs present in all non-LTR trypanosomatid elements, similar to the C2H2 zinc finger family of transcription factors. Interestingly, ORF1 codes for a protein with significant homology to the major human AP endonuclease protein, and maintains in similar positions most of the amino acid domains described for all the Ape family of proteins. The presence of Ape-related sequences, described for the first time in a non-LTR retrotransposon (L1Tc), may have functional relevance for these types of elements.

Amino Acid Sequence↗
PubMed1994

Generality of the tRNA origin of short interspersed repetitive elements (SINEs). Characterization of three different tRNA-derived retroposons in the octopus.

Transcription in vitro of total genomic DNA of Octopus vulgaris resulted in PolIII transcripts of 200 to 500 nucleotides in length. These transcripts were used as probes and, as a result, three different kinds of short interspersed element (SINE) were isolated and characterized. Two SINEs, designated the octopus OR1 and OR2 families, seem to have been derived from tRNA(Arg). The other SINE, designated the octopus OK family, have originated from tRNA, but the parental tRNA species cannot be identified due to sequence divergence from the original tRNA sequence. The OR1 and OR2 families exhibit considerable similarity to one another, in the 5' region of the tRNA-unrelated region as well as in the tRNA(Arg)-related region, an observation that suggests that these two families may have had the same origin in evolution. The three SINEs together constitute at least 6% of the genome of O. vulgaris. Results of a dot hybridization experiment suggest that the OR1 and OR2 families are present in fewer species than the OK family and that the OK family exists in many species of octopodid. The present observations indicate that SINEs have generally been derived from tRNAs in invertebrates, as well as in vertebrates, and that retroposition is widely involved in the genetic and structural variability of invertebrate genomes. It has been demonstrated that tRNA(Arg) and tRNA(Lys) appear to be tRNA species found as progenitors of vertebrate SINEs, and the same preference for progenitor species of tRNAs seems also to be a feature of invertebrate SINEs.

Animals↗
PubMed1995

Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition.

Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.

Base Sequence↗
PubMed1997

Ribonuclease and high salt sensitivity of the ribonucleoprotein complex formed by the human LINE-1 retrotransposon.

P40 is encoded by the first open reading frame of the human LINE-1 retrotransposon and is found in a large cytoplasmic ribonucleoprotein (RNP) complex, the p40 RNP-complex, in association with LINE-1 RNA(s) in human teratocarcinoma cell lines. We report here investigations on the stability of the p40 RNP-complex against various nucleases and high salt (0.5 M NaCl) treatment. The results indicate that (1) the p40 RNP-complex is dissociated after ribonuclease or high salt treatment, (2) DNase I does not disrupt the complex, (3) after dissociation of the complex, p40 maintain protein-protein interactions but in smaller complexes, and (4) p40 is not associated with the LINE-1 RNA(s) after high salt treatment. These observations suggest that the RNA molecule(s) is(are) essential for the stability of the large p40 complex and that the complex has a structure which allows various nucleases to reach the RNA. These features are distinct from those of typical virus and virus-like particles of retroviruses and other retrotransposons, respectively. Together with the fact that no significant sequence homology exists between p40 and the gag and gag-like proteins, it is likely that the p40 RNP-complex is structurally different from the typical virus and virus-like particles.

Blotting, Western↗
PubMed1997

De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence.

The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.

Base Composition↗
PubMed1998

Recombination between subtypes creates a mosaic lineage of LINE-1 that is expressed and actively retrotransposing in the mouse genome.

LINE-1, or L1, elements are retrotransposons that have amplified to high-copy number during the evolution of mammals. L1 appears to amplify in waves, spawning large numbers of progeny such that elements with distinct sequence features dominate the dispersal process in a given window of time. This process generates discrete subfamilies of L1 within mammalian genomes, with the oldest being remnants, or fossils, of earlier waves of amplification. In mice, at least three distinct subfamilies of L1 were distinguished by their unique 5' ends, A, F and V. These subfamilies amplified at distinct times in the evolution of mice, with A being the youngest and V the oldest; both V and F subfamilies were believed extinct. Recent data established that a variant of the F family, TF, is actively retrotransposing. We demonstrate here that members of the TF subfamily are abundantly expressed in mouse cells and encode the major protein constituent of L1 ribonucleoprotein particles. Although members of the TF subfamily are not as numerous in the genomes of laboratory mice as are members of the older A and F subfamilies, they appear to have been activated some time ago during mouse evolution, in the common ancestor of Mus spretus and Mus domesticus. Phylogenetic analysis demonstrates that this modern, active form of TF-type L1 has a composite evolutionary history, showing evidence of multiple recombinations between distinct L1 variants, including members of the A and F subfamilies.

Animals↗
PubMed1998

High-resolution cartography of recently integrated human chromosome 19-specific Alu fossils.

The recently inserted subfamilies of Alu retroposons (Ya5/8 and Yb8) are composed of approximately 2000 elements. We have screened a human chromosome 19-specific cosmid library for the presence of Ya5/8 and Yb8 Alu family members. This analysis resulted in the identification of 12 Ya5/8 Alu family members and 15 Yb8 Alu family members from human chromosome 19. The total number of Ya5/8 and Yb8 Alu family members located on human chromosome 19 does not differ from that expected based upon random integration of Alu repeats within the human genome. The distribution of both subfamilies of Alu elements along human chromosome 19 also appears to be random. DNA sequence analysis of the individual Alu elements revealed a low level of random mutations within both subfamilies of Alu elements consistent with their recent evolutionary origin. Oligonucleotide primers complementary to the flanking unique sequences adjacent to each Alu element were used in polymerase chain reaction assays to determine the phylogenetic distribution and human genomic variation associated with each Alu family member. All of the chromosome 19-specific Ya5/8 and Yb8 Alu family members were restricted to the human genome and absent from orthologous positions within the genomes of several non-human primates. Three of the Yb8 Alu family members were polymorphic for insertion presence/absence within the genomes of a diverse array of human populations. The polymorphic Alu elements will be useful tools for the study of human population genetics.

Animals↗
PubMed1999

A second gene encoding the mouse proteasome activator PA28beta subunit is part of a LINE1 element and is driven by a LINE1 promoter.

The PMSE2 gene encodes the beta-subunit of the proteasome activator PA28 and, as shown by genomic Southern blot analysis, there probably exist four copies sharing sequence homology with PMSE2. Here, we report that in the mouse genome there exist two different chromosomal loci for PA28beta, both of which are transcribed and and which encode a functional PA28beta subunit. One of these represents the previously described PMSE2 gene possessing an intron-exon structure and a gamma interferon (IFNgamma)-inducible promoter. The second one, named PMSE2b, which we describe here, exhibits all the characteristics of an expressed retrotransposon. Our data show that the PA28beta retrotransposon is inserted into a transcriptional active LINE1 element and is driven by a LINE1 F-type monomer promoter as revealed by luciferase assays. The resulting PMSE2b mRNA encodes a protein which is indistinguishable from that encoded by the IFNgamma-inducible PMSE2 gene. Since PA28 plays an important role in major histocompatibility complex class I antigen presentation, the implications for the mouse immune system through a constitutively expressed PA28beta subunit and the biological relevance of this finding are discussed.

Animals↗
PubMed1999

Reverse transcription of the yeast Ty1 retrotransposon: the mode of first strand transfer is either intermolecular or intramolecular.

Replication of the yeast Ty1 retrotransposon occurs by a mechanism similar to that of retroviruses. According to the current model of retroviral reverse transcription, two strand transfers (the so-called minus-strand and plus-strand strong-stop DNA transfers) are required to produce full-length preintegrative DNA. Because two genomic RNA molecules are packaged inside the viral particles, the strand transfers can be either intra- or intermolecular. To study the mode of transfer of minus-strand strong-stop DNA during reverse transcription of the yeast Ty1 retrotransposon, we have analyzed the cDNA products that accumulate in the cytoplasmic virus-like particles of yeast cells harboring two marked Ty1 elements. Our results indicate that Ty1 minus-strand transfer occurs in a random manner with approximately similar frequencies of intra- and intermolecular transfer. It has been observed recently that intra- and intermolecular minus-strand transfer occur at similar frequencies during replication of a complex retrovirus such as HIV-1. These results together with the observation that genetic recombination occurs with a high frequency during minus-strand synthesis suggest that both packaged RNA molecules are needed for the synthesis of one minus-strand DNA.

Base Sequence↗
PubMed1999

Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein.

The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores. Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein. The VLPs are highly polydisperse in their radius distribution. We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles. Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles. This assembly property provides a model for understanding the variable assembly of retroviral core proteins. The particles are assembled from trimer-clustered units and there are holes in the capsid shells.

Capsid↗
PubMed2000

Upstream flanking sequences and transcription of SINEs.

SINEs, short interspersed repeated DNA elements, undergo amplification through retroposition and subsequent integration into a new location in the genome. Each new SINE insertion will be located in a new chromosomal environment, with different flanking sequences. Modulation of transcription by different flanking sequences may play an important role in determining which SINE elements are preferentially active in a genome. We evaluated the ability of upstream flanking sequences to regulate the transcription of three different SINEs (Alu, B2 and ID) by constructing chimeric constructs with known 5' flanking sequences of RNA polymerase III-transcribed genes. Upstream sequences from the 7SL RNA gene, U6 RNA gene, vault RNA gene, and BC1 gene increase transcription of Alu, B2 and BC1 in transient transfections of NIH3T3, HeLa, Neuro2a and C6 glioma cell lines. The 7SL sequence proved most efficient in increasing SINE transcription. The 7SL upstream fused to the BC1 RNA gene (an ID element) was used to create a transgenic mouse line. In contrast to the tissue-specific endogenous BC1 transcription, BC1 transgene transcripts were detected in all tissues tested. However, expression was much higher in those tissues that express the endogenous gene, demonstrating both transcriptional and post-transcriptional regulation. The BC1 RNA was detected in a similar ribonucleoprotein complex in the different tissues.

Alu Elements↗
PubMed2002

The reverse transcriptase of the R2 non-LTR retrotransposon: continuous synthesis of cDNA on non-continuous RNA templates.

R2 is a non-long terminal repeat (non-LTR) retrotransposon that inserts into the 28 S rRNA genes of arthropods. The element encodes two enzymatic activities: an endonuclease that specifically cleaves the 28 S gene target site, and a reverse transcriptase (RT) that can use the 3' end of the cleaved DNA to prime reverse transcription. R2 RT only utilizes RNA templates that contain the 3' untranslated region of the R2 element as templates in this target primed reverse transcription (TPRT) reaction. Here, detailed biochemical characterization of the R2 RT indicates that the enzyme is capable of making multiple, consecutive jumps between RNA templates. The terminal 3' nucleotide of the "acceptor" RNA and the 5' nucleotide of the "donor" RNA are frequently reverse transcribed in these jumps, indicating that the acceptor RNA does not anneal to the cDNA derived from the donor RNA template. These template jumps occur during TPRT as well as in non-specific extension reactions in which reverse transcription is primed by an oligonucleotide annealed to the RNA template. Analysis of these RT assays done in the absence of the target DNA also revealed that the R2 RT can initiate reverse transcription near the 3' end of any RNA molecule using the 3' end of a second RNA molecule as primer. Again there is no requirement for sequence complementarity between the RNA used as template and the RNA used as primer. These properties of the R2 RT differ substantially from those of retroviral RTs but have similarities to the RT of the Mauriceville retroplasmid of Neurospora crassa. We present a model which relates these unusual properties of the R2 RT to structural differences from retroviral RTs as well as correlates these properties to the likely retrotransposition mechanism of R2 and other non-LTR retrotransposons.

Animals↗