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Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes.

Bilanchone V, Clemens K, Kaake R, Dawson AR, Matheos D, Nagashima K, Sitlani P, Patterson K, Chang I, Huang L, Sandmeyer S - PLoS Genet. (2015)

Bottom Line: Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified.These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition.Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Irvine, Irvine, California, United States of America.

ABSTRACT
Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.

No MeSH data available.


Related in: MedlinePlus

Xrn1 and Lsm1 are required for Gag3 foci formation in mating cells.(A) Localization of Ty3 Gag3 and RNA in WT (BY4741) and lsm1Δ or xrn1Δ strains. Cells were imaged by confocal microscopy after 4 h of α-factor induction. Ty3 Gag3 was detected by immunofluorescence using antibody against Ty3 VLPs and Ty3 RNA was detected by FISH using Ty3-specific oligonucleotides (S1 Text, Supporting Information Methods and Materials). Insert indicates % cells with RNA foci (mean ± SD). Scale bar = 2 μm. (B) Gag3-ΔNC fails to localize to PB foci. Ty3Δ (yVB1672) cells were transformed with plasmids expressing either WT Ty3 (pTD3685) or with Ty3 with deletion of the NC domain of Gag3 (NCΔ) (pPS3705) under the native promoter. Cells were imaged by confocal microscopy after 4 h of α-factor induction as described in (A). Polysome analysis of WT (BY4741) or xrn1Δ cells induced with α-factor for 2 h. Cell lysates, either untreated (C) or treated (D) with EDTA, were analyzed by sucrose gradient sedimentation. The 40, 60 and 80s ribosomal subunits and polysomes were monitored with continuous A260 measurements. Total RNA was extracted from even number fractions and was analyzed by northern blot for Ty3 (green) and ACT1 RNA (orange). Protein was extracted from odd number fractions and was analyzed by western blotting for Gag3 (red) and CA (pink). RNA and protein data is expressed as a percentage of the total in all fractions (distribution %). Data are representative of results from two independent experiments.
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pgen.1005528.g007: Xrn1 and Lsm1 are required for Gag3 foci formation in mating cells.(A) Localization of Ty3 Gag3 and RNA in WT (BY4741) and lsm1Δ or xrn1Δ strains. Cells were imaged by confocal microscopy after 4 h of α-factor induction. Ty3 Gag3 was detected by immunofluorescence using antibody against Ty3 VLPs and Ty3 RNA was detected by FISH using Ty3-specific oligonucleotides (S1 Text, Supporting Information Methods and Materials). Insert indicates % cells with RNA foci (mean ± SD). Scale bar = 2 μm. (B) Gag3-ΔNC fails to localize to PB foci. Ty3Δ (yVB1672) cells were transformed with plasmids expressing either WT Ty3 (pTD3685) or with Ty3 with deletion of the NC domain of Gag3 (NCΔ) (pPS3705) under the native promoter. Cells were imaged by confocal microscopy after 4 h of α-factor induction as described in (A). Polysome analysis of WT (BY4741) or xrn1Δ cells induced with α-factor for 2 h. Cell lysates, either untreated (C) or treated (D) with EDTA, were analyzed by sucrose gradient sedimentation. The 40, 60 and 80s ribosomal subunits and polysomes were monitored with continuous A260 measurements. Total RNA was extracted from even number fractions and was analyzed by northern blot for Ty3 (green) and ACT1 RNA (orange). Protein was extracted from odd number fractions and was analyzed by western blotting for Gag3 (red) and CA (pink). RNA and protein data is expressed as a percentage of the total in all fractions (distribution %). Data are representative of results from two independent experiments.

Mentions: The lsm1Δ and xrn1Δ strains showed increased amounts of Gag3, but this contrasted with decreased processing in xrn1Δ cells and decreased packaging of Ty3 gRNA in both mutants. In addition, the Ty3-GFP reporter showed deficient focus formation in lsm1Δ and xrn1Δ cells (lsm1Δ = 15±8% and xrn1Δ = 38±5%) (Fig 3). These properties suggested a defect in transitioning the gRNA from translation into assembly phases or in assembly itself. Because of programmed frameshifting between GAG3 and POL3, Gag3 is about 20-fold more abundant than Gag3-Pol3 [63]. To increase the sensitivity of analysis, cells were induced with pheromone for Ty3 expression for 8 h and Gag3 was analyzed by immunofluorescence using an anti-VLP antibody that reacts with Gag3 (see Materials and Methods). RNA was detected by fluorescence in situ hybridization (FISH) using three pooled oligonucleotide probes complementary to Ty3 internal regions. WT cells showed strong co-localization of Gag3 and Ty3 RNA in cytoplasmic foci (Fig 7A). Analysis of lsm1Δ and xrn1Δ strains showed a punctate cytoplasmic signal interspersed with fewer intense foci than in WT cells (lsm1Δ = 55±3% and xrn1Δ = 35±6%, compared to WT = 71±5%). However, the overall faintness of the RNA signal prevented a determination as to whether the Ty3 RNA in the mutants also formed fewer foci. RNA foci that were observed were co-localized with Gag3 puncta. These observations suggested that Gag3 retained interaction with Ty3 gRNA, but failed to coalesce normally into assembly foci. This interpretation was confirmed by comparison of this phenotype to that of a Ty3 Gag3-NCΔ mutant, lacking most of the NC-coding region so that it fails to interact with gRNA. This mutant failed to form Gag3 foci and did not have detectable co-localization of Gag3 and gRNA [11](Fig 7B). We conclude that despite an abundance of Ty3 RNA and Gag3, lsm1Δ and xrn1Δ strains fail to efficiently form Ty3-PB foci so that protein and RNA remain diffuse.


Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes.

Bilanchone V, Clemens K, Kaake R, Dawson AR, Matheos D, Nagashima K, Sitlani P, Patterson K, Chang I, Huang L, Sandmeyer S - PLoS Genet. (2015)

Xrn1 and Lsm1 are required for Gag3 foci formation in mating cells.(A) Localization of Ty3 Gag3 and RNA in WT (BY4741) and lsm1Δ or xrn1Δ strains. Cells were imaged by confocal microscopy after 4 h of α-factor induction. Ty3 Gag3 was detected by immunofluorescence using antibody against Ty3 VLPs and Ty3 RNA was detected by FISH using Ty3-specific oligonucleotides (S1 Text, Supporting Information Methods and Materials). Insert indicates % cells with RNA foci (mean ± SD). Scale bar = 2 μm. (B) Gag3-ΔNC fails to localize to PB foci. Ty3Δ (yVB1672) cells were transformed with plasmids expressing either WT Ty3 (pTD3685) or with Ty3 with deletion of the NC domain of Gag3 (NCΔ) (pPS3705) under the native promoter. Cells were imaged by confocal microscopy after 4 h of α-factor induction as described in (A). Polysome analysis of WT (BY4741) or xrn1Δ cells induced with α-factor for 2 h. Cell lysates, either untreated (C) or treated (D) with EDTA, were analyzed by sucrose gradient sedimentation. The 40, 60 and 80s ribosomal subunits and polysomes were monitored with continuous A260 measurements. Total RNA was extracted from even number fractions and was analyzed by northern blot for Ty3 (green) and ACT1 RNA (orange). Protein was extracted from odd number fractions and was analyzed by western blotting for Gag3 (red) and CA (pink). RNA and protein data is expressed as a percentage of the total in all fractions (distribution %). Data are representative of results from two independent experiments.
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pgen.1005528.g007: Xrn1 and Lsm1 are required for Gag3 foci formation in mating cells.(A) Localization of Ty3 Gag3 and RNA in WT (BY4741) and lsm1Δ or xrn1Δ strains. Cells were imaged by confocal microscopy after 4 h of α-factor induction. Ty3 Gag3 was detected by immunofluorescence using antibody against Ty3 VLPs and Ty3 RNA was detected by FISH using Ty3-specific oligonucleotides (S1 Text, Supporting Information Methods and Materials). Insert indicates % cells with RNA foci (mean ± SD). Scale bar = 2 μm. (B) Gag3-ΔNC fails to localize to PB foci. Ty3Δ (yVB1672) cells were transformed with plasmids expressing either WT Ty3 (pTD3685) or with Ty3 with deletion of the NC domain of Gag3 (NCΔ) (pPS3705) under the native promoter. Cells were imaged by confocal microscopy after 4 h of α-factor induction as described in (A). Polysome analysis of WT (BY4741) or xrn1Δ cells induced with α-factor for 2 h. Cell lysates, either untreated (C) or treated (D) with EDTA, were analyzed by sucrose gradient sedimentation. The 40, 60 and 80s ribosomal subunits and polysomes were monitored with continuous A260 measurements. Total RNA was extracted from even number fractions and was analyzed by northern blot for Ty3 (green) and ACT1 RNA (orange). Protein was extracted from odd number fractions and was analyzed by western blotting for Gag3 (red) and CA (pink). RNA and protein data is expressed as a percentage of the total in all fractions (distribution %). Data are representative of results from two independent experiments.
Mentions: The lsm1Δ and xrn1Δ strains showed increased amounts of Gag3, but this contrasted with decreased processing in xrn1Δ cells and decreased packaging of Ty3 gRNA in both mutants. In addition, the Ty3-GFP reporter showed deficient focus formation in lsm1Δ and xrn1Δ cells (lsm1Δ = 15±8% and xrn1Δ = 38±5%) (Fig 3). These properties suggested a defect in transitioning the gRNA from translation into assembly phases or in assembly itself. Because of programmed frameshifting between GAG3 and POL3, Gag3 is about 20-fold more abundant than Gag3-Pol3 [63]. To increase the sensitivity of analysis, cells were induced with pheromone for Ty3 expression for 8 h and Gag3 was analyzed by immunofluorescence using an anti-VLP antibody that reacts with Gag3 (see Materials and Methods). RNA was detected by fluorescence in situ hybridization (FISH) using three pooled oligonucleotide probes complementary to Ty3 internal regions. WT cells showed strong co-localization of Gag3 and Ty3 RNA in cytoplasmic foci (Fig 7A). Analysis of lsm1Δ and xrn1Δ strains showed a punctate cytoplasmic signal interspersed with fewer intense foci than in WT cells (lsm1Δ = 55±3% and xrn1Δ = 35±6%, compared to WT = 71±5%). However, the overall faintness of the RNA signal prevented a determination as to whether the Ty3 RNA in the mutants also formed fewer foci. RNA foci that were observed were co-localized with Gag3 puncta. These observations suggested that Gag3 retained interaction with Ty3 gRNA, but failed to coalesce normally into assembly foci. This interpretation was confirmed by comparison of this phenotype to that of a Ty3 Gag3-NCΔ mutant, lacking most of the NC-coding region so that it fails to interact with gRNA. This mutant failed to form Gag3 foci and did not have detectable co-localization of Gag3 and gRNA [11](Fig 7B). We conclude that despite an abundance of Ty3 RNA and Gag3, lsm1Δ and xrn1Δ strains fail to efficiently form Ty3-PB foci so that protein and RNA remain diffuse.

Bottom Line: Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified.These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition.Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Irvine, Irvine, California, United States of America.

ABSTRACT
Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.

No MeSH data available.


Related in: MedlinePlus