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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

Time course of pheromone-induced production of Ty3 retrotransposition intermediates.(A) Gag3 RNA levels determined by RT-qPCR. (B) Quantification of Gag3 protein product levels and Gag3 processing determined by immunoblot analysis using α-CA antibody and α-Pgk1 antibody as control. (C) Ty3 cDNA levels determined by Southern blot analysis and endogenous element fragment as control. (D) Ty3 assembly intermediates identified in α-factor pulse-chase experiment by fractionation of cell extracts by velocity sedimentation. Cell culture was induced by α-factor for 1.5 h (top panel) or followed by a 4 h chase (bottom panels). Proteins were extracted from gradient fractions and analyzed for Ty3 proteins by immunoblot, DNA by Southern blot and RNA by northern blot. (S1 Text, Supporting Materials and Methods). (E) TEM image of Ty3 VLPs located adjacent to a nuclear pore after induction by α-factor for 6 h. n, nucleus; np, nuclear pore; arrow, position of three-fold enlarged image (inset). Scale bar = 500 nm.
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pgen.1005528.g001: Time course of pheromone-induced production of Ty3 retrotransposition intermediates.(A) Gag3 RNA levels determined by RT-qPCR. (B) Quantification of Gag3 protein product levels and Gag3 processing determined by immunoblot analysis using α-CA antibody and α-Pgk1 antibody as control. (C) Ty3 cDNA levels determined by Southern blot analysis and endogenous element fragment as control. (D) Ty3 assembly intermediates identified in α-factor pulse-chase experiment by fractionation of cell extracts by velocity sedimentation. Cell culture was induced by α-factor for 1.5 h (top panel) or followed by a 4 h chase (bottom panels). Proteins were extracted from gradient fractions and analyzed for Ty3 proteins by immunoblot, DNA by Southern blot and RNA by northern blot. (S1 Text, Supporting Materials and Methods). (E) TEM image of Ty3 VLPs located adjacent to a nuclear pore after induction by α-factor for 6 h. n, nucleus; np, nuclear pore; arrow, position of three-fold enlarged image (inset). Scale bar = 500 nm.

Mentions: To establish the timeframe of assembly of Ty3 VLPs and cDNA synthesis in cells undergoing pheromone induction, haploid yeast strain MATa BY4741 (Table 1), that contains two endogenous Ty3 elements (yGRWTy3-1 and yILWTy3-1), was exposed to pheromone from MATα cells (α-factor) for 2 h (Fig 1). Levels of the 5.2-kb Ty3 gRNA increase dramatically between 0 and 2 h of induction (Fig 1A) [14]. Gag3 polyprotein precursor was detected by 2 h and continued to accumulate up to the 8 h sampling. The Gag3-Pol3 fusion product of frameshifting is made at about one-twentieth the level of Gag [63] and was not detected under these experimental conditions. Gag3-Pol3 contains the PR domain. Upon VLP formation the Ty3 aspartyl protease, PR, is activated to convert Gag3 into CA, spacer and NC, and Gag3-Pol3 additionally into PR, RT, and IN species [59]. Appearance of these forms is consistent with substantial formation of VLPs between 4 and 6 h [9] (Fig 1B). Ty3 VLP maturation occurs in G1, but cDNA synthesis is delayed until after cells undergo fusion to form diploid zygotes and resume the cell cycle in S phase [60]. Between 6 and 8 h, haploid cells recover from pheromone arrest and cDNA synthesis is substantial (Fig 1C).


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)

Time course of pheromone-induced production of Ty3 retrotransposition intermediates.(A) Gag3 RNA levels determined by RT-qPCR. (B) Quantification of Gag3 protein product levels and Gag3 processing determined by immunoblot analysis using α-CA antibody and α-Pgk1 antibody as control. (C) Ty3 cDNA levels determined by Southern blot analysis and endogenous element fragment as control. (D) Ty3 assembly intermediates identified in α-factor pulse-chase experiment by fractionation of cell extracts by velocity sedimentation. Cell culture was induced by α-factor for 1.5 h (top panel) or followed by a 4 h chase (bottom panels). Proteins were extracted from gradient fractions and analyzed for Ty3 proteins by immunoblot, DNA by Southern blot and RNA by northern blot. (S1 Text, Supporting Materials and Methods). (E) TEM image of Ty3 VLPs located adjacent to a nuclear pore after induction by α-factor for 6 h. n, nucleus; np, nuclear pore; arrow, position of three-fold enlarged image (inset). Scale bar = 500 nm.
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pgen.1005528.g001: Time course of pheromone-induced production of Ty3 retrotransposition intermediates.(A) Gag3 RNA levels determined by RT-qPCR. (B) Quantification of Gag3 protein product levels and Gag3 processing determined by immunoblot analysis using α-CA antibody and α-Pgk1 antibody as control. (C) Ty3 cDNA levels determined by Southern blot analysis and endogenous element fragment as control. (D) Ty3 assembly intermediates identified in α-factor pulse-chase experiment by fractionation of cell extracts by velocity sedimentation. Cell culture was induced by α-factor for 1.5 h (top panel) or followed by a 4 h chase (bottom panels). Proteins were extracted from gradient fractions and analyzed for Ty3 proteins by immunoblot, DNA by Southern blot and RNA by northern blot. (S1 Text, Supporting Materials and Methods). (E) TEM image of Ty3 VLPs located adjacent to a nuclear pore after induction by α-factor for 6 h. n, nucleus; np, nuclear pore; arrow, position of three-fold enlarged image (inset). Scale bar = 500 nm.
Mentions: To establish the timeframe of assembly of Ty3 VLPs and cDNA synthesis in cells undergoing pheromone induction, haploid yeast strain MATa BY4741 (Table 1), that contains two endogenous Ty3 elements (yGRWTy3-1 and yILWTy3-1), was exposed to pheromone from MATα cells (α-factor) for 2 h (Fig 1). Levels of the 5.2-kb Ty3 gRNA increase dramatically between 0 and 2 h of induction (Fig 1A) [14]. Gag3 polyprotein precursor was detected by 2 h and continued to accumulate up to the 8 h sampling. The Gag3-Pol3 fusion product of frameshifting is made at about one-twentieth the level of Gag [63] and was not detected under these experimental conditions. Gag3-Pol3 contains the PR domain. Upon VLP formation the Ty3 aspartyl protease, PR, is activated to convert Gag3 into CA, spacer and NC, and Gag3-Pol3 additionally into PR, RT, and IN species [59]. Appearance of these forms is consistent with substantial formation of VLPs between 4 and 6 h [9] (Fig 1B). Ty3 VLP maturation occurs in G1, but cDNA synthesis is delayed until after cells undergo fusion to form diploid zygotes and resume the cell cycle in S phase [60]. Between 6 and 8 h, haploid cells recover from pheromone arrest and cDNA synthesis is substantial (Fig 1C).

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