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Thomas H Eickbush

Publications and source records attributed to Thomas H Eickbush.

At least 19 recordsLinked to original sources
PubMed2006

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

5' Untranslated Regions↗
PubMed2006

Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of Drosophila melanogaster.

About half of the rRNA gene units (rDNA units) of Drosophila melanogaster are inserted by the retrotransposable elements R1 and R2. Because transcripts to R1 and R2 were difficult to detect on blots and electron microscopic observations of rRNA synthesis suggested that only uninserted rDNA units were transcribed, it has long been postulated that inserted rDNA units are in a repressed (inactive) chromatin structure. Studies described here suggest that inserted and uninserted units are equally accessible to DNase I and micrococcal nuclease and contain similar levels of histone H3 and H4 acetylation and H3K9 methylation. These studies have low sensitivity, because psoralen cross-linking suggested few (estimated <10%) of the rDNA units of any type are transcriptionally active. Nuclear run-on experiments revealed that R1-inserted and R2-inserted units are activated for transcription at about 1/5 and 1/10, respectively, the rate of uninserted units. Most transcription complexes of the inserted units terminate within the elements, thus explaining why previous molecular and electron microscopic methods indicated inserted units are seldom transcribed. The accumulating data suggest that all units within small regions of the rDNA loci are activated for transcription, with most control over R1 and R2 activity involving steps downstream of transcription initiation.

Animals↗
PubMed2005

Role of the Bombyx mori R2 element N-terminal domain in the target-primed reverse transcription (TPRT) reaction.

R2 is a site-specific non-long terminal repeat (non-LTR) retrotransposon encoding a single polypeptide with reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The current model of R2 retrotransposition involves an ordered series of cleavage and polymerization steps carried out by at least two R2 protein subunits, one bound upstream and one bound downstream of the integration site. The role in the retrotransposition reaction of two conserved DNA-binding motifs, a C2H2 zinc finger (ZF) and a Myb motif, located within the N-terminal domain of the protein are explored in this report. These motifs do not appear to play a role in RT or the ability of the protein to bind the R2 RNA transcript. Methylation and missing nucleoside interference-based DNA footprints using polypeptides to the N-terminal domain suggest the ZF and Myb motifs bind to regions -3 to -1 and +10 to +15 with reference to the insertion site. Mutations in these DNA sites or of the N-terminal protein domain blocked binding and the activity of the downstream subunit. Mutations of the protein domain also affected binding of the upstream subunit but not its function, suggesting the primary path to DNA target recognition by R2 involves both upstream and downstream subunits.

Amino Acid Motifs↗
PubMed2005

Monitoring the mode and tempo of concerted evolution in the Drosophila melanogaster rDNA locus.

Non-LTR retrotransposons R1 and R2 have persisted in rRNA gene loci (rDNA) since the origin of arthropods despite their continued elimination by the recombinational mechanisms of concerted evolution. This study evaluated the short-term evolutionary dynamics of the rDNA locus by measuring the divergence among replicate Drosophila melanogaster lines after 400 generations. The total number of rDNA units on the X chromosome of each line varied from 140 to 310, while the fraction of units inserted with R1 and R2 retrotransposons ranged from 37 to 65%. This level of variation is comparable to that found in natural population surveys. Variation in locus size and retrotransposon load was correlated with large changes in the number of uninserted and R1-inserted units, yet the numbers of R2-inserted units were relatively unchanged. Intergenic spacer (IGS) region length variants were also used to evaluate changes in the rDNA loci. All IGS length variants present in the lines showed significant increases and decreases of copy number. These studies, combined with previous data following specific R1 and R2 insertions in these lines, help to define the type and distribution, both within the locus and within the individual units, of recombinational events that give rise to the concerted evolution of the rDNA locus.

Animals↗
PubMed2005

Characterization of active R2 retrotransposition in the rDNA locus of Drosophila simulans.

The rRNA gene (rDNA) loci of all arthropod lineages contain non-LTR retrotransposable elements that have evolved to specifically insert into the 28S rRNA genes. Extensive in vitro experiments have been conducted to investigate the mechanism of R2 retrotransposition but little is known of the insertion frequency or cellular factors that might regulate R2 activity. In this article, isofemale lines obtained from a population of Drosophila simulans were surveyed for recent R2 insertions. Within most lines, all individuals showed the same collection of R2 insertions, providing no evidence for recent R2 activity. However, in a few of the isofemale lines, virtually all individuals differed in their R2 insertion profiles. The descendants of individual pairs of flies from these "active lines" rapidly accumulated new insertions. The frequent insertion of new R2 elements was associated with the elimination of old R2 elements from the rDNA locus. The existence of lines in which R2 retrotransposes frequently and lines in which the elements appear dormant suggests that cellular mechanisms that can regulate the activity of R2 exist. Retrotransposition activity was correlated with the number of full-length R2 elements but not with the size of the rDNA locus or the number of uninserted units.

Animals↗
PubMed2005

R2 target-primed reverse transcription: ordered cleavage and polymerization steps by protein subunits asymmetrically bound to the target DNA.

R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.

Animals↗
PubMed2004

Footprint of the retrotransposon R2Bm protein on its target site before and after cleavage.

R2 elements are non-long terminal repeat (non-LTR) retrotransposons that specifically integrate into the 28 S rRNA genes of their host. These elements encode a single open reading frame with a genome-specific endonuclease and a reverse transcriptase that uses the cleaved chromosomal target site to prime reverse transcription. Cleavage of the DNA strand that is used to prime reverse transcription is an efficient process that occurs in the presence or absence of RNA. Cleavage of the second DNA strand is much less efficient and requires RNA. Reverse transcription occurs before second strand cleavage and only if the RNA bound to the protein contains the 3' untranslated region of the R2 element. Thus a complex series of protein interactions with the DNA and conformational changes in the protein are likely to occur during this retrotransposition reaction. Here, we conduct electrophoretic mobility-shift assays and DNase I footprint studies on the binding of the R2 protein to the DNA target in the presence and absence of RNA both before and after first strand cleavage. While the total expanse of the protein footprint on the DNA eventually covers five helical turns, before cleavage the footprint only extends from 17 bp to 40 bp upstream of the cleavage site. This footprint is the same in the presence and absence of RNA. We hypothesize that the active site of the endonuclease domain is analogous to type IIS restriction enzymes in that it is located on a flexible domain that is not tightly bound to the cleavage site. After first strand cleavage the protein footprint extends beyond the cleavage site. We suggest that this increased protection after cleavage is the RT domain that is positioned over the free DNA end to begin reverse transcription on the nicked DNA substrate.

Animals↗
PubMed2004

End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon.

The reverse transcriptase encoded by the non-long terminal repeat retrotransposon R2 has been shown to be able to jump from the 5'-end of one RNA template (the donor) to the 3'-end of a second RNA template (the acceptor) in the absence of preexisting sequence identity between the two templates. These jumps between RNA templates have similarity to the end-to-end template jumps described for the RNA-directed RNA polymerases encoded by certain RNA viruses. Here we describe for the first time the mechanism by which such end-to-end template jumps can occur. Most template jumps by the R2 reverse transcriptase are brought about by the enzyme's ability to add nontemplated (overhanging) nucleotides to the cDNA when it reaches the end of the donor RNA. The enzyme then anneals these overhanging nucleotides to sequences at the 3'-end of the acceptor RNA. The annealing is most efficient if it involves the terminal nucleotide(s) of the acceptor RNA but can occur to sites at least 5 nucleotides from the 3'-end. These end-to-end jumps are similar to steps proposed to be part of the integration reaction of non-long terminal repeat retrotransposons and can explain chimeric integration products derived from multiple RNA templates.

Cross-Linking Reagents↗
PubMed2004

Secondary structure models of the 3' untranslated regions of diverse R2 RNAs.

The RNA structure of the 3' untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure-function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3' UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3' UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with beta-ethoxy-alpha-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function.

3' Untranslated Regions↗
PubMed2003

R5 retrotransposons insert into a family of infrequently transcribed 28S rRNA genes of planaria.

A small (100 bp) region of the 28S rRNA gene has been shown to serve as the target site for the insertion of non-long terminal repeat (non-LTR) retrotransposons in both arthropods and nematodes. Here we characterize a lineage of non-LTR retrotransposons that inserts into this target site in the phylum Platyhelminthes. Dugesiid planaria contain elements, named R5, that insert 8 bp upstream of the target site used by arthropod R2 elements. The complete sequence of this element from Girardia tigrina revealed that it encoded two open reading frames (ORFs). The second ORF contained reverse transcriptase and restriction enzyme-like endonuclease domains similar to those found in R2 and R4, the elements that insert into the 28S genes of nematodes. The closest relative of R5, however, was the element NeSL-1, which inserts into the spliced leader 1 exons of nematodes. The rRNA genes of dugesiid planaria are unusual in that they comprise two types of rDNA units that differ by 8%-10% in nucleotide sequence of the 18S and 28S coding regions. Type II units are transcribed in adult tissues at levels that are less than 1% that of the type I units. R5 elements were only found inserted in the type II units, where presumably they cause less harm to the host. A second unusual aspect of the dugesiid rRNA genes is that the target site for the R5 insertion is duplicated 300 bp upstream of the original insertion site. R5 elements were identified at both sites. These findings expand the distribution of non-LTR elements that are specialized for insertion into the 28S gene and suggest that still more elements exist in other eukaryotic taxa. Attempts to trace the phylogeny of R5 did not offer sufficient resolution to determine whether R2, R4, and R5 represent the same lineage or whether they represent independent specializations for the 28S gene.

Animals↗
PubMed2003

R1 and R2 retrotransposition and deletion in the rDNA loci on the X and Y chromosomes of Drosophila melanogaster.

The non-LTR retrotransposons R1 and R2 insert into the 28S rRNA genes of arthropods. Comparisons among Drosophila lineages have shown that these elements are vertically inherited, while studies within species have indicated a rapid turnover of individual copies (elimination of old copies and the insertion of new copies). To better understand the turnover of R1 and R2, 200 retrotranspositions and nearly 100 eliminations have been scored in the Harwich mutation-accumulation lines of Drosophila melanogaster. Because the rDNA arrays in D. melanogaster are present on the X and Y chromosomes and no exchanges were detected in these lines, it was possible to show that R1 retrotranspositions occur predominantly in the male germ line, while R2 retrotranspositions were more evenly divided between the germ lines of both sexes. The rate of elimination of elements from the Y rDNA array was twice that of the X rDNA array with both chromosomal loci containing regions where the rate of elimination was on average eight times higher. Most R1 and R2 eliminations appear to occur by large intrachromosomal events (i.e., loop-out events) that involve multiple rDNA units. These findings are interpreted in light of the known abundance of R1 and R2 elements in the X and Y rDNA loci of D. melanogaster.

Animals↗
PubMed2003

Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of Drosophila melanogaster.

R2 retrotransposons insert into the rRNA-encoding units (rDNA units) that form the nucleoli of insects. We have utilized an R2 integration system in Drosophila melanogaster to study transcription of foreign sequences integrated into the R2 target site of the 28S rRNA genes. The exogenous sequences were cotranscribed at dramatically different levels which closely paralleled the level of transcription of the endogenous R1 and R2 elements. Transcription levels were inversely correlated with the number of uninserted rDNA units, variation in this number having been brought about by the R2 integration system itself. Females with as few as 20 uninserted rDNA units per X chromosome had expression levels of endogenous and exogenous insertion sequences that were 2 orders of magnitude higher than lines that contained over 80 uninserted rDNA units per chromosome. R2 insertions only 167 bp in length exhibited this range of transcriptional regulation. Analysis of transcript levels in males suggested R2 insertions on the Y chromosome are not down-regulated to the same extent as insertions on the X chromosome. These results suggest that transcription of the rDNA units can be tightly regulated, but this regulation gradually breaks down as the cell approaches the minimum number of uninserted genes needed for survival.

Animals↗
PubMed2002

R2 retrotransposition on assembled nucleosomes depends on the translational position of the target site.

R2 retrotransposons insert into the 28S rRNA genes of insects. Integration occurs by specific cleavage of the target site and utilization of the released DNA end to prime reverse transcription of the RNA transcript. Specificity of the protein to the target site is dependent upon nucleotide sequence recognition extending from 35 bp upstream to 15 bp downstream of the cleavage site. In this report, we show that sequence recognition and cleavage by the R2 protein can occur while the target site is assembled into nucleosomes. Reconstitution of DNA fragments containing the 28S gene sequence into a set of nucleosomes with different translational frames revealed that the R2 site adopted the same rotational orientation with respect to the histone octamer. Binding and cleavage by the R2 protein were most efficient when the upstream binding site for the R2 protein was near a nucleosome end. Interaction of the R2 protein with the nucleosome disrupted the histone:DNA contacts in the 50 bp region directly bound by R2, but did not modify the remainder of the nucleosome structure.

Animals↗
PubMed2002

High processivity of the reverse transcriptase from a non-long terminal repeat retrotransposon.

R2 is a retrotransposable element that specifically inserts into the 28 S rRNA genes of arthropods. The element encodes a single protein with endonuclease activity that cleaves the 28 S gene target site and reverse transcriptase (RT) activity that uses the cleaved DNA to prime reverse transcription. Here we compare various properties of the R2 RT activity with those of the well characterized retroviral RT, avian myeloblastosis virus (AMV). In processivity assays using heterogeneous RNA templates, R2 RT can synthesize cDNA over twice the length of that synthesized by AMV RT and can synthesize cDNA over 4 times longer than AMV RT in assays with poly(rA) templates. The higher processivity of R2 RT compared with retroviral RTs is a result of the slower rate of dissociation of the enzyme from RNA templates. The elongation rates of the two enzymes are similar. Finally, a highly distinct property of the R2 RT, compared with retroviral enzymes, is its ability to displace RNA strands annealed to RNA templates during cDNA synthesis. We suggest that both the higher processivity and displacement properties of R2 RT compared with retroviral RT result from the greater affinity of the R2 protein for the RNA template upstream of its active site.

Avian Myeloblastosis Virus↗
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↗
PubMed2002

Fruit flies and humans respond differently to retrotransposons.

Retrotransposable element insertions are 20 times more numerous per unit length of DNA in the large human genome compared to the small Drosophila genome. Whereas all Drosophila elements are subject to constant turnover (recent insertion and elimination by selection), this has not generally been the case for human retrotransposons. We suggest that a difference in recombination adopted by these organisms in response to the deleterious effects of interspersed repeated DNA can explain in part this fundamental difference between the evolutionary dynamics of fruit fly and human retrotransposons.

Animals↗
PubMed2002

Rates of R1 and R2 retrotransposition and elimination from the rDNA locus of Drosophila melanogaster.

R1 and R2 elements are non-LTR retrotransposons that insert specifically into the 28S rRNA genes of arthropods. The process of concerted evolution of the rDNA locus should give rise to rapid turnover of these mobile elements compared to elements that insert at sites throughout a genome. To estimate the rate of R1 and R2 turnover we have examined the insertion of new elements and elimination of old elements in the Harwich mutation accumulation lines of Drosophila melanogaster, a set of inbred lines maintained for >350 generations. Nearly 300 new insertion and elimination events were observed in the 19 Harwich lines. The retrotransposition rate for R1 was 18 times higher than the retrotransposition rate for R2. Both rates were within the range previously found for retrotransposons that insert outside the rDNA loci in D. melanogaster. The elimination rates of R1 and R2 from the rDNA locus were similar to each other but over two orders of magnitude higher than that found for other retrotransposons. The high rates of R1 and R2 elimination from the rDNA locus confirm that these elements must maintain relatively high rates of retrotransposition to ensure their continued presence in this locus.

Animals↗