A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposon under tight transcriptional control.

A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription

ABSTRACTBeyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our studies expand our understanding of the complex interactions between retrotransposons and the NPC, and highlight the importance for the cells to keep retrotransposon under tight transcriptional control.AUTHOR SUMMARYRetroelements, which replicate by reverse transcription of their RNA into a cDNA that is integrated into the host genetic material, play an important role in the plasticity of eukaryotic genomes. The study of yeast retrotransposons has led to the identification of host factors that limit retroelement mobility, including components of the nuclear pore complex (NPC), most of them still awaiting mechanistic characterization. Here, we investigated the contribution of the Nup84 complex, a core NPC scaffold, to retrotransposon biology in budding yeast. Our findings uncover that the Nup84 complex restricts the transcription of phylogenetically-distinct Ty retroelements. By focusing on Ty1 retrotransposons, we provide evidence that repression by the Nup84 complex depends on the maintenance at the NPC of the SUMO-protease Ulp1, an essential enzyme of the SUMO pathway with multiple targets in the transcription machinery. We finally show that this transcriptional control is critical for genome dynamics, since a small increase in the accumulation of Ty1 RNAs leads to massive retrotransposition. Our data reveal that although relatively abundant, Ty transcripts are limiting for retrotransposition, underscoring the importance of a tight control of their expression. They also characterize a new non-canonical function of NPCs, confirming their connection with genome expression and stability.

Oxidal® Ameliorates the Ty1 Retrotransposition Induced by Methyl Methanesulfonate in Saccharomyces cerevisiae

Aim: The aim of the study was to evaluate the potential of Oxidal® to decrease the Ty1 retrotransposition rate in a model system Saccharomyces cerevisiae. Study Design: Saccharomyces cerevisiae cell suspensions were pre-treated with different concentrations Oxidal® and subsequently treated with 16mM methyl methanesulfonate. (MMS) Methodology: The potential of various concentrations Oxidal® was evaluated based on “spot” test and Ty1 retro-transposition test. Results: Data revealed that only 5% Oxidal® possesses some cytotoxic properties. Lack of Ty1 retro-transposition was observed after single treatment with 1, 2.5 and 5% Oxidal® concentrations. On the other hand, all the tested concentrations showed promising results against the standard carcinogen methyl methane sulfonate. The most pronounced anti-carcinogenic and cytoprotective effects were observed after pre-treatment with 2.5% Oxidal®, which could be attributed to the antioxidant properties of the combination of ingredients; methylene blue, salicylic acid and caffeine. Further studies could reveal the exact mechanism of action of the supplement and the role of the antioxidant potential. Conclusion: New data is provided concerning the potential of Oxidal® at low concentrations to protect Saccharomyces cerevisiae cells from MMS-induced Ty1 retro-transposition. The cytoprotective properties of the supplement were also obtained. These results could be considered as a basis for further studies revealing the exact mechanisms of cell protection of the Oxidal®. Additionally, our data could also serve as an important step of the in-depth research of a potential antiviral activity.

The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting nonessential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility alter a post-translational step(s), Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.Author SummaryRetrotransposons are mobile genetic elements that copy their RNA genomes into DNA and insert the DNA copies into the host genome. These elements contribute to genome instability, control of host gene expression and adaptation to changing environments. Retrotransposons depend on numerous host factors for their own propagation and control. The retrovirus-like retrotransposon, Ty1, in the yeast Saccharomyces cerevisiae has been an invaluable model for retrotransposon research, and hundreds of host factors that regulate Ty1 retrotransposition have been identified. Non-essential subunits of the Mediator transcriptional co-activator complex have been identified as one set of host factors implicated in Ty1 regulation. Here, we report a systematic investigation of the effects of loss of these non-essential subunits of Mediator on Ty1 retrotransposition. Our findings reveal a heretofore unknown mechanism by which Mediator influences the balance between transcription from two promoters in Ty1 to modulate expression of an autoinhibitory transcript known as Ty1i RNA. Our results provide new insights into host control of retrotransposon activity via promoter choice and elucidate a novel mechanism by which the Mediator co-activator governs this choice.

Atrans-Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

ABSTRACTSaccharomyces cerevisiaeandSaccharomyces paradoxuslack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. GlutathioneS-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation.IMPORTANCERetrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both “host and parasite.” To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate retroelement replication.