Thermal blockage of viruslike particle formation for the yeast retrotransposon Ty3 reveals differences in the cellular stress response

Sadeghi, Naseem; Rütz, Marie-Louise; Menees, Thomas M.

doi:10.1007/s007050170042

Cited by 10 publications

(11 citation statements)

References 27 publications

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“…A quantitative version of this assay showed that the frequency of transposition in the wild-type strain was threefold that of the rad52 strain (data not shown). This was similar to the twofold difference between wildtype and rad52 derivative previously reported (Sadeghi et al 2001). Enrichment for cells that had undergone transposition before production of genomic libraries for sequencing was necessitated by the low percentage of transformants (<1%) that undergo transposition and by the diversity of those events.…”

Section: Resultssupporting

confidence: 59%

Retrotransposon profiling of RNA polymerase III initiation sites

Qi¹,

Daily²,

Nguyen³

et al. 2012

Genome Res.

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Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes.

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Section: Resultssupporting

confidence: 59%

Retrotransposon profiling of RNA polymerase III initiation sites

Qi¹,

Daily²,

Nguyen³

et al. 2012

Genome Res.

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“…Replica-plating assays have been described previously (15,29,35). Briefly, patches of cells containing pGAL1-Ty1 are replica plated to medium containing 2% D-(ϩ)-galactose to induce transcription of Ty1.…”

Section: Methodsmentioning

confidence: 99%

Relationship between RNA Lariat Debranching and Ty1 Element Retrotransposition

Salem

Boucher

Menees

2003

J Virol

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The Saccharomyces cerevisiae DBR1 gene encodes a 2-5 phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5 end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2-5 phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.Saccharomyces cerevisiae Ty1 is a long terminal repeat (LTR) retroelement with a life cycle similar to that of retroviruses (2). Like retroviruses, Ty1 replicates via reverse transcription of an RNA intermediate, subsequently integrating element cDNA into the host genome. Retroviruses and LTR retrotransposons rely on their hosts to provide most of the factors necessary for propagation. We identified dbr1 mutants in a screen to identify host cell genes required for Ty1 transposition (15). The dbr1 mutant had been identified previously in a similar screen by others (6). Although dbr1 cells produce wild-type levels of Ty1 proteins, they accumulate Ty1 cDNA at a slower rate than do wild-type cells, suggesting that Dbr1p plays a role in Ty1 reverse transcription or cDNA stability (15).In its cellular role, Dbr1p acts at the end of the mRNA splicing process, cleaving the 2Ј-5Ј linkage at the branch point of intron RNA lariats released after exon ligation, converting them to linear RNAs that are rapidly degraded (6, 23). In a dbr1 mutant, intron RNAs accumulate as lariats (6). The uncleaved lariat branch point blocks the progression of 3Ј exonucleases, and there is no 5Ј end available on which a 5Ј exonuclease can act. DBR1 is highly conserved, and homologues have been cloned from Schizosaccharomyces pombe, S. cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (6,17,18,24). Deletion of DBR1 has no significant effect on the growth rate of S. cerevisiae, but...

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“…Construction of a high-copy, URA3-marked plasmid, pNB2142, which carries a GAL1-10 UAS -regulated, HIS3-marked Ty3 element (Ty3-HIS3) derived from a HIS3AI-marked element (Sadeghi et al 2001) has been described (Aye et al 2004). In order to make a low-copy, Ty3-HIS3 expression plasmid, the GAL1-10 UAS and Ty3 in pNB2142 was amplified by PCR and cloned between the HindIII and Not1 sites of the low-copy, URA3 plasmid pRS316.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

Retroviruses and yeast retrotransposons use overlapping sets of host genes

Irwin¹,

Aye²,

Baldi³

et al. 2005

Genome Res.

127

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A collection of 4457 Saccharomyces cerevisiae mutants deleted for nonessential genes was screened for mutants with increased or decreased mobilization of the gypsylike retroelement Ty3. Of these, 64 exhibited increased and 66 decreased Ty3 transposition compared with the parental strain. Genes identified in this screen were grouped according to function by using GOnet software developed as part of this study. Gene clusters were related to chromatin and transcript elongation, translation and cytoplasmic RNA processing, vesicular trafficking, nuclear transport, and DNA maintenance. Sixty-six of the mutants were tested for Ty3 proteins and cDNA. Ty3 cDNA and transposition were increased in mutants affected in nuclear pore biogenesis and in a subset of mutants lacking proteins that interact physically or genetically with a replication clamp loader. Our results suggest that nuclear entry is linked mechanistically to Ty3 cDNA synthesis but that host replication factors antagonize Ty3 replication. Some of the factors we identified have been previously shown to affect Ty1 transposition and others to affect retroviral budding. Host factors, such as these, shared by distantly related Ty retroelements and retroviruses are novel candidates for antiviral targets.

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Thermal blockage of viruslike particle formation for the yeast retrotransposon Ty3 reveals differences in the cellular stress response

Cited by 10 publications

References 27 publications

Retrotransposon profiling of RNA polymerase III initiation sites

Retrotransposon profiling of RNA polymerase III initiation sites

Relationship between RNA Lariat Debranching and Ty1 Element Retrotransposition

Retroviruses and yeast retrotransposons use overlapping sets of host genes

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