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Ty3, a Position-specific Retrotransposon in Budding Yeast

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  • Authors: Suzanne Sandmeyer1, Kurt Patterson3, Virginia Bilanchone4
  • Editors: Suzanne Sandmeyer5, Nancy Craig6
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Biological Chemistry; 2: Department of Microbiology and Molecular Genetics, University of California, Irvine, CA; 3: Department of Biological Chemistry; 4: Department of Biological Chemistry; 5: University of California, Irvine, CA; 6: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014
  • Received 22 January 2015 Accepted 30 January 2015 Published 19 March 2015
  • Suzanne Sandmeyer, sbsandme@uci.edu
image of Ty3, a Position-specific Retrotransposon in Budding Yeast
  • Abstract:

    Long terminal repeat (LTR) retrotransposons constitute significant fractions of many eukaryotic genomes. Two ancient families are Ty1/Copia () and Ty3/Gypsy (). The Ty3/Gypsy family probably gave rise to retroviruses based on the domain order, similarity of sequences, and the envelopes encoded by some members. The Ty3 element of is one of the most completely characterized elements at the molecular level. Ty3 is induced in mating cells by pheromone stimulation of the mitogen-activated protein kinase pathway as cells accumulate in G1. The two Ty3 open reading frames are translated into Gag3 and Gag3–Pol3 polyprotein precursors. In haploid mating cells Gag3 and Gag3–Pol3 are assembled together with Ty3 genomic RNA into immature virus-like particles in cellular foci containing RNA processing body proteins. Virus-like particle Gag3 is then processed by Ty3 protease into capsid, spacer, and nucleocapsid, and Gag3–Pol3 into those proteins and additionally, protease, reverse transcriptase, and integrase. After haploid cells mate and become diploid, genomic RNA is reverse transcribed into cDNA. Ty3 integration complexes interact with components of the RNA polymerase III transcription complex resulting in Ty3 integration precisely at the transcription start site. Ty3 activation during mating enables proliferation of Ty3 between genomes and has intriguing parallels with metazoan retrotransposon activation in germ cell lineages. Identification of nuclear pore, DNA replication, transcription, and repair host factors that affect retrotransposition has provided insights into how hosts and retrotransposons interact to balance genome stability and plasticity.

  • Citation: Sandmeyer S, Patterson K, Bilanchone V. 2015. Ty3, a Position-specific Retrotransposon in Budding Yeast. Microbiol Spectrum 3(2):MDNA3-0057-2014. doi:10.1128/microbiolspec.MDNA3-0057-2014.

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/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0057-2014
2015-03-19
2018-07-16

Abstract:

Long terminal repeat (LTR) retrotransposons constitute significant fractions of many eukaryotic genomes. Two ancient families are Ty1/Copia () and Ty3/Gypsy (). The Ty3/Gypsy family probably gave rise to retroviruses based on the domain order, similarity of sequences, and the envelopes encoded by some members. The Ty3 element of is one of the most completely characterized elements at the molecular level. Ty3 is induced in mating cells by pheromone stimulation of the mitogen-activated protein kinase pathway as cells accumulate in G1. The two Ty3 open reading frames are translated into Gag3 and Gag3–Pol3 polyprotein precursors. In haploid mating cells Gag3 and Gag3–Pol3 are assembled together with Ty3 genomic RNA into immature virus-like particles in cellular foci containing RNA processing body proteins. Virus-like particle Gag3 is then processed by Ty3 protease into capsid, spacer, and nucleocapsid, and Gag3–Pol3 into those proteins and additionally, protease, reverse transcriptase, and integrase. After haploid cells mate and become diploid, genomic RNA is reverse transcribed into cDNA. Ty3 integration complexes interact with components of the RNA polymerase III transcription complex resulting in Ty3 integration precisely at the transcription start site. Ty3 activation during mating enables proliferation of Ty3 between genomes and has intriguing parallels with metazoan retrotransposon activation in germ cell lineages. Identification of nuclear pore, DNA replication, transcription, and repair host factors that affect retrotransposition has provided insights into how hosts and retrotransposons interact to balance genome stability and plasticity.

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FIGURE 1

Ty3 retrotransposition. (A) Ty3 replication cycle. Pheromone binding to or pheromone receptors activates G protein-coupled mating signal transduction via mitogen-activated protein (MAP) kinase kinase kinase Ste7, MAP kinase kinase Ste11 and MAP kinase Fus3 (rose). Scaffold protein Ste5 (blue) supports specificity of their interaction preventing crosstalk with the filamentous growth pathway. Fus3 phosphorylates Dig1 and Dig2 negative regulators (gold) of Ste12 (dark blue), which then dissociate allowing Ste12 activation of RNA Pol II transcription of Ty3. Ty3 poly(A) RNA (maroon) is exported and translated into Gag3 and Gag3–Pol3 (tan), which then associate, together with the gRNA and RNA processing body (PB) factors, forming retrosomes within which Ty3 VLPs assemble. These foci become perinuclear over time. Assembly activates protease (PR) processing and maturation of the virus-like particles (VLPs). After cells mate (not shown) reverse transcription of the gRNA into cDNA occurs. Uncoating (dissociation of Gag3) presumably accompanies nuclear entry of the PIC. (A, B) Ty3 cDNA associates with RNAP III transcription initiation complexes composed of TFIIIB (yellow) and TFIIIC (green). TATA binding protein and Brf1 constitute the minimum target, but evidence suggests that TFIIIC can also be present. doi:10.1128/microbiolspec.MDNA3-0057-2014.f1

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014
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FIGURE 2a

Ty3 DNA, RNA, and protein. (A) DNA and RNA. The 5.4-kbp Ty3 element is transcribed into a 5.2-kb RNA. The major 5′ end of Ty3 RNA maps to 118 nucleotides (nt) inside the upstream long terminal repeat (LTR), and the 3′ end to heterogeneous positions between 243 and 273 nt inside the downstream LTR, as well as beyond the downstream LTR ( 14 ). The overlap resulting from termination downstream of the position of initiation results in a sequence that is repeated (“R”) and defines 5′ U5 and 3′ U3 sequences. The initiator AUG of the open reading frame (ORF) occurs at nucleotides 76 to 78 inside the Ty3 internal domain for a total 5′ untranslated region (UTR) of 193 nt; extends into the downstream LTR to define a 3′ UTR of ∼227 nt ( 12 , 14 ). Candidate upstream TATA and downstream polyadenylation sites are identified, but not experimentally verified. The RNA contains a bipartite primer binding site (PBS), which anneals to initiator tRNA in the upstream untranslated region and the downstream LTR (gold boxes). The and ORFs encode Gag3 and Gag3–Pol3. (B) Protein. Gag3 is 290 amino acids (aa) and contains domains that mature via Ty3 PR processing into 207-aa capsid (CA), 27-aa spacer (SP), and 57-aa nucleocapsid (NC). Gag3–Pol3 contains those and additionally, protease (PR); reverse transcriptase (RT) starting at amino acids 536; and two forms of integrase (IN) domains (starting at amino acids 1012 and 1038) produced via a programmed frameshift. The ORF terminates within the downstream LTR so that the polypurine tract (PPT) plus strand primer is actually within the IN-coding region. (C) Reverse transcription of Ty3 genomic RNA. The tRNA primes synthesis of a minus-strand strong stop containing U5 and R segments, which then transfers to the 3′ end and primes extension of the minus strand. The plus-strand strong stop intermediate is initiated with cleavage by RNaseH at the downstream end of the PPT just outside the downstream LTR and is extended through U3, R, and U5 and likely copied into the 3′ end of the tRNA then transferred to the 5′ end of the RNA and extended to form the plus strand of the cDNA. Although as described in the text minus- and plus-strand strong-stop intermediates have been identified, the overall flow described is based on the retrovirus model. An additional possibility (not shown) is that the 5′ and 3′ ends are transiently joined in a lariat RNA (see text). Bottom, the full-length cDNA has two extra base pairs on each end derived from a 2-nt offset between the priming sites and the LTR ends of the integrated element. Integrase (IN) processes 2 nt from each 3′ end and mediates the nucleophilic attacks of the resulting hydroxyls at 5-bp staggered positions flanking the RNA polymerase III (RNAPIII) transcription initiation sites. The integration site is repaired, resulting in 5-bp direct repeats flanking the ends of the Ty3 element. doi:10.1128/microbiolspec.MDNA3-0057-2014.f2a

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014
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FIGURE 2b

Ty3 DNA, RNA, and protein. (A) DNA and RNA. The 5.4-kbp Ty3 element is transcribed into a 5.2-kb RNA. The major 5′ end of Ty3 RNA maps to 118 nucleotides (nt) inside the upstream long terminal repeat (LTR), and the 3′ end to heterogeneous positions between 243 and 273 nt inside the downstream LTR, as well as beyond the downstream LTR ( 14 ). The overlap resulting from termination downstream of the position of initiation results in a sequence that is repeated (“R”) and defines 5′ U5 and 3′ U3 sequences. The initiator AUG of the open reading frame (ORF) occurs at nucleotides 76 to 78 inside the Ty3 internal domain for a total 5′ untranslated region (UTR) of 193 nt; extends into the downstream LTR to define a 3′ UTR of ∼227 nt ( 12 , 14 ). Candidate upstream TATA and downstream polyadenylation sites are identified, but not experimentally verified. The RNA contains a bipartite primer binding site (PBS), which anneals to initiator tRNA in the upstream untranslated region and the downstream LTR (gold boxes). The and ORFs encode Gag3 and Gag3–Pol3. (B) Protein. Gag3 is 290 amino acids (aa) and contains domains that mature via Ty3 PR processing into 207-aa capsid (CA), 27-aa spacer (SP), and 57-aa nucleocapsid (NC). Gag3–Pol3 contains those and additionally, protease (PR); reverse transcriptase (RT) starting at amino acids 536; and two forms of integrase (IN) domains (starting at amino acids 1012 and 1038) produced via a programmed frameshift. The ORF terminates within the downstream LTR so that the polypurine tract (PPT) plus strand primer is actually within the IN-coding region. (C) Reverse transcription of Ty3 genomic RNA. The tRNA primes synthesis of a minus-strand strong stop containing U5 and R segments, which then transfers to the 3′ end and primes extension of the minus strand. The plus-strand strong stop intermediate is initiated with cleavage by RNaseH at the downstream end of the PPT just outside the downstream LTR and is extended through U3, R, and U5 and likely copied into the 3′ end of the tRNA then transferred to the 5′ end of the RNA and extended to form the plus strand of the cDNA. Although as described in the text minus- and plus-strand strong-stop intermediates have been identified, the overall flow described is based on the retrovirus model. An additional possibility (not shown) is that the 5′ and 3′ ends are transiently joined in a lariat RNA (see text). Bottom, the full-length cDNA has two extra base pairs on each end derived from a 2-nt offset between the priming sites and the LTR ends of the integrated element. Integrase (IN) processes 2 nt from each 3′ end and mediates the nucleophilic attacks of the resulting hydroxyls at 5-bp staggered positions flanking the RNA polymerase III (RNAPIII) transcription initiation sites. The integration site is repaired, resulting in 5-bp direct repeats flanking the ends of the Ty3 element. doi:10.1128/microbiolspec.MDNA3-0057-2014.f2b

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014
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FIGURE 3

Retrotransposition assays. Ty3 retrotransposition can be assayed using the reporter embedded in the mobilized Ty3 (genetic) or by PCR. In the case of the Genetic Assay (left panel), Ty3 transcription is accompanied by splicing of a synthetic intron which is antisense to the marker, preventing from being productively expressed. After Ty3 transcription, splicing and reverse transcription, the intronless gene is expressed and cells in which transposition has occurred are selected on medium lacking histidine. Alternatively, retrotransposition of a tagged Ty3 element can be monitored by PCR assay (right panel) using one primer complementary to the Ty3 tag and one complementary to a sequence present in a tDNA family or in the unique sequence downstream of any tDNA target. doi:10.1128/microbiolspec.MDNA3-0057-2014.f3

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014
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FIGURE 4

Hierarchical clustering of Ty3 cofactors and restriction factors by gene ontology groups. Gene ontology (GO) analysis was performed using the GO SLIM Biological Process mapping tool available through Genome Database (http://www.yeastgenome.org). Knockout mutants identified as having either increased “Up” or decreased “Down” Ty3 retrotransposition phenotypes were analyzed for GO: Biological Process terms. Enriched categories were determined using chi-squared test. GO categories were considered enriched if two criteria were met: (i) the -value was <0.05 and (ii) the number of genes in the enriched category exceeded 10% of the total number of genes in the Up or Down list. Enriched categories were converted to a heat map with hierarchical clustering using R; values represent the [–Log(-value)] scaled from 0 (no significance) to 1, blue coloring reflects the intensity of significance. doi:10.1128/microbiolspec.MDNA3-0057-2014.f4

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014
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Tables

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TABLE 1

Host factors in common between Ty3 and HIV-1

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0057-2014

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