Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements - PubMed (original) (raw)
Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements
J Yang et al. Proc Natl Acad Sci U S A. 1999.
Abstract
The non-long terminal repeat (LTR) retrotransposon, R2, encodes a sequence-specific endonuclease responsible for its insertion at a unique site in the 28S rRNA genes of arthropods. Although most non-LTR retrotransposons encode an apurinic-like endonuclease upstream of a common reverse transcriptase domain, R2 and many other site-specific non-LTR elements do not (CRE1 and 2, SLACS, CZAR, Dong, R4). Sequence comparison of these site-specific elements has revealed that the region downstream of their reverse transcriptase domain is conserved and shares sequence features with various prokaryotic restriction endonucleases. In particular, these non-LTR elements have a Lys/Arg-Pro-Asp-X12-14aa-Asp/Glu motif known to lie near the scissile phosphodiester bonds in the protein-DNA complexes of restriction enzymes. Site-directed mutagenesis of the R2 protein was used to provide evidence that this motif is also part of the active site of the endonuclease encoded by this element. Mutations of this motif eliminate both DNA-cleavage activities of the R2 protein: first-strand cleavage in which the exposed 3' end is used to prime reverse transcription of the RNA template and second-strand cleavage, which occurs after reverse transcription. The general organization of the R2 protein appears similar to the type IIS restriction enzyme, FokI, in which specific DNA binding is controlled by a separate domain located amino terminal to the cleavage domain. Previous phylogenetic analysis of their reverse transcriptase domains has indicated that the non-LTR elements identified here as containing restriction-like endonucleases are the oldest lineages of non-LTR elements, suggesting a scenario for the evolution of non-LTR elements.
Figures
Figure 1
Identification of the putative endonuclease domain in R2 and other site-specific non-LTR elements. (A) Schematic diagram of the R2 ORF from B. mori and its comparison to the C-terminal ends of other site-specific non-LTR retrotransposable elements. Shaded regions in R2 indicate the RT domain, putative zinc finger (CCHH), and c-myb-like DNA-binding motifs. Carboxyl terminal to the RT domain in all elements is a putative zinc finger motif (CCHC) and a motif (PD..D) with similarity to restriction enzymes. (B) Sequence comparison of the putative endonuclease domain of nine R2 elements from diverse arthropods (9) with those of other sequence-specific non-LTR elements and with some restriction endonucleases. The number of amino acid residues between the conserved motifs is given in parentheses. Highly conserved residues are shown in shaded boxes, with the three charged residues that are part of the active site of the restriction enzymes also bolded. The region between these conserved residues assumes a β-turn in restriction enzymes (24, 25). The large number of charged residues in these β-turns are indicated as light gray.
Figure 2
Enzymatic activity of the R2 protein mutations. (A) TPRT assay by using a 110-bp 5′ end-labeled target. The two strands of the target DNA are represented by the straight lines, with the labeled 5′ ends noted with an asterisk. The cDNA made in the reaction is indicated by a straight line, and the RNA template is indicated by a dashed line. The 283-nt RNA template contains the sequence of the 3′ untranslated region of the silkmoth R2 element. In the absence of TPRT, 60- and 48-nt labeled DNA fragments are detected on a denaturing gel. If TPRT occurs, a 343-nt fragment also is generated. (B) Autoradiography of the TPRT reaction run on an 8% denaturing polyacrylamide gel. For each reaction, 15 ng (200 fmol) of end-labeled DNA and 4 ng (30 fmol) of protein were incubated for 30 min. Lanes: 1, no protein; 2, wild-type R2 protein; 3, PA..D mutation; 4, PE..D mutation; and 5, YAYD mutation. Numbers to the right indicate the lengths of the observed DNA products.
Figure 3
Endonuclease mutants can conduct the TPRT reaction on prenicked DNA substrates. (A) Schematic diagram summarizing the substrates and products of the TPRT reactions. The two strands of the DNA substrate are indicated by thin lines; the cDNA synthesized during the reaction is indicated with a thicker line and the RNA template is indicated with a wavy line. (B) Autoradiographs of the reaction products with the PA..D mutation (Left) and PE..D mutation (Right) separated on 1% agarose gels. Only plasmids that have undergone the TPRT reaction can be seen in these autoradiograms. For each reaction, 0.25 μg supercoiled or prenicked plasmid DNA was incubated with the R2 protein as in Fig. 2, except that 2 μCi [α-32P]dATP was added to each reaction. Lanes: 1 and 5, no protein controls; 2–4, 4, 8, and 16 ng of the mutant protein incubated with the supercoiled target; 6–8, 4, 8, and 16 ng of the mutant protein incubated with the prenicked target; and 9, 16 ng of wild-type protein incubated with supercoiled DNA.
Figure 4
Complementation of the endonuclease and RT mutations. Assays were performed as diagrammed in Fig. 2_A_, by using the 110-bp end-labeled DNA target. Lanes: 1, no protein; 2, 4 ng PE..D mutation; 3, 4 ng PE..D and 4 ng YAYD mutations; and 4, 4 ng YAYD mutation. Neither the PE..D or YAYD mutant alone can use the DNA target to prime reverse transcription of the 283-nt R2 RNA; however, a mixture of the two proteins is capable. Twice the total level of protein was added in lane 3 compared with the wild-type lane in Fig. 2 to enable the formation of an equivalent amount of an active heterodimer of PE..D and YAYD compared with a wild-type dimer. We have no direct evidence, however, that such a heterodimer is formed.
Figure 5
Comparison of the ORFs encoded by group II introns and non-LTR retrotransposable elements. The group II intron shown is the ltrB intron of Lactococcus lactis (37). The protein domains shared by other group II introns are shaded and are similar to those identified previously (41), except that the domains referred to as Z and X in that study are shown here as part of the RT domain (see ref. 8). The putative endonuclease domain of the group II introns is identified as HNH (38, 39). In the case of the non-LTR retrotransposons, schematic diagrams of the R2, L1, and Jockey elements are shown as representatives of the major non-LTR structures found to date. Other major lineages of non-LTR elements with these basic structures are listed within the parentheses (8). The CCHH, c-myb, CCHC, and PD..D domains of the R2 elements are described in Fig. 1. The AP-like endonuclease domain identified at the amino-terminal end of L1 and Jockey elements is labeled APE. Elements with structures similar to L1 contain a CCHC domain downstream of their RT domain; thus, this region is likely to be involved in DNA binding. Arrows represent the likely path of non-LTR evolution in eukaryotes based on the phylogeny of their RT domains (8).
Similar articles
- The domain structure and retrotransposition mechanism of R2 elements are conserved throughout arthropods.
Burke WD, Malik HS, Jones JP, Eickbush TH. Burke WD, et al. Mol Biol Evol. 1999 Apr;16(4):502-11. doi: 10.1093/oxfordjournals.molbev.a026132. Mol Biol Evol. 1999. PMID: 10331276 - R4, a non-LTR retrotransposon specific to the large subunit rRNA genes of nematodes.
Burke WD, Müller F, Eickbush TH. Burke WD, et al. Nucleic Acids Res. 1995 Nov 25;23(22):4628-34. doi: 10.1093/nar/23.22.4628. Nucleic Acids Res. 1995. PMID: 8524653 Free PMC article. - Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.
Luan DD, Eickbush TH. Luan DD, et al. Mol Cell Biol. 1996 Sep;16(9):4726-34. doi: 10.1128/MCB.16.9.4726. Mol Cell Biol. 1996. PMID: 8756630 Free PMC article. - Integration, Regulation, and Long-Term Stability of R2 Retrotransposons.
Eickbush TH, Eickbush DG. Eickbush TH, et al. Microbiol Spectr. 2015 Apr;3(2):MDNA3-0011-2014. doi: 10.1128/microbiolspec.MDNA3-0011-2014. Microbiol Spectr. 2015. PMID: 26104703 Free PMC article. Review. - Transposing without ends: the non-LTR retrotransposable elements.
Eickbush TH. Eickbush TH. New Biol. 1992 May;4(5):430-40. New Biol. 1992. PMID: 1325183 Review.
Cited by
- CRISPR technologies for genome, epigenome and transcriptome editing.
Villiger L, Joung J, Koblan L, Weissman J, Abudayyeh OO, Gootenberg JS. Villiger L, et al. Nat Rev Mol Cell Biol. 2024 Jun;25(6):464-487. doi: 10.1038/s41580-023-00697-6. Epub 2024 Feb 2. Nat Rev Mol Cell Biol. 2024. PMID: 38308006 Review. - rDNA magnification is a unique feature of germline stem cells.
Nelson JO, Kumon T, Yamashita YM. Nelson JO, et al. Proc Natl Acad Sci U S A. 2023 Nov 21;120(47):e2314440120. doi: 10.1073/pnas.2314440120. Epub 2023 Nov 15. Proc Natl Acad Sci U S A. 2023. PMID: 37967216 Free PMC article. - The retrotransposon R2 maintains Drosophila ribosomal DNA repeats.
Nelson JO, Slicko A, Yamashita YM. Nelson JO, et al. Proc Natl Acad Sci U S A. 2023 Jun 6;120(23):e2221613120. doi: 10.1073/pnas.2221613120. Epub 2023 May 30. Proc Natl Acad Sci U S A. 2023. PMID: 37252996 Free PMC article. - Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription.
Wilkinson ME, Frangieh CJ, Macrae RK, Zhang F. Wilkinson ME, et al. Science. 2023 Apr 21;380(6642):301-308. doi: 10.1126/science.adg7883. Epub 2023 Apr 6. Science. 2023. PMID: 37023171 Free PMC article. - Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon.
Fefelova EA, Pleshakova IM, Mikhaleva EA, Pirogov SA, Poltorachenko VA, Abramov YA, Romashin DD, Shatskikh AS, Blokh RS, Gvozdev VA, Klenov MS. Fefelova EA, et al. Nucleic Acids Res. 2022 Jan 25;50(2):867-884. doi: 10.1093/nar/gkab1276. Nucleic Acids Res. 2022. PMID: 35037046 Free PMC article.
References
- Eickbush T H. In: The Evolutionary Biology of Viruses. Morse S S, editor. New York: Raven; 1994. pp. 121–157.
- Whitcomb J M, Hughes S H. Annu Rev Cell Biol. 1992;8:275–306. - PubMed
- Mizuuchi K. Annu Rev Biochem. 1992;61:1011–1051. - PubMed
- Luan D D, Korman M H, Jakubczak J L, Eickbush T H. Cell. 1993;72:595–605. - PubMed
- Martin F, Maranon C, Olivares M, Alonso C, Lopez M C. J Mol Biol. 1995;247:49–59. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases