Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages

Nf1 RasGAP inhibition of LIMK2 mediates a new cross-talk between Ras and Rho pathways.

BACKGROUND: Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity: the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition: in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator. CONCLUSIONS/SIGNIFICANCE: Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases

SecPH partially inhibits cofilin phosphorylation by LIMK2, but not by LIMK1.

<p><i>A</i>. <i>Actin cytoskeleton organisation</i>. HeLa cells were cotransfected with pcDNA3, LIMK2 and SecPH or its parental empty plasmid, p3XFlag. Cells were fixed and stained with phalloidin, or anti-HA or anti-flag antibodies. <i>B. Inhibition of LIMK2 cofilin phosphorylation by SecPH.</i> Cells were cotransfected with LIMK2 and SecPH or Galectin-3 (a non-specific control protein). Immunoprecipitated HA-LIMK2 and GST-cofilin were used in the kinase assay. The kinase activity on cofilin of immunoprecipitated HA-LIMK2 from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. Statistical significance was determined relative to control using one-way ANOVA (* p<0.05). The HA-immunoprecipitates were also submitted to HA-immunoblotting and to coomassie blue staining. Lysates were submitted to flag-immunoblotting. <i>C. Dose dependent inhibition of LIMK2 cofilin phosphorylation by SecPH.</i> Cells were transfected with LIMK2 and either SecPH or Galectin-3. SecPH and Galectin-3 cell lysates were immunoprecipitated with anti-flag beads, beads were then eluted flag peptide. Immunoprecipitated HA-LIMK2 and GST-cofilin were used for kinase assay and were incubated with increasing amount of immunoprecipitated SecPH or Galectin-3 (0, 6, 12, 18 ul respectively). The kinase activity on cofilin of immunoprecipitated HA-LIMK2 with no addition of immunoprecipitated SecPH or Galectin-3 was taken as 1.0. Each value represents the mean ± SE of four independent experiments. Statistical significance was determined relative to control using one-way ANOVA (*** p<0.0001). Immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining. <i>D. SecPH does not inhibit cofilin phosphorylation by LIMK1.</i> Cells were transfected either with SecPH or with LIMK1. SecPH cell lysates were immunoprecipitated with anti-flag beads, beads were then eluted with flag peptide. Immunoprecipitated HA-LIMK1 was used for kinase assay and was incubated with increasing amount of immunoprecipitated SecPH (0, 6, 12, 18 ul respectively). The kinase activity on cofilin of immunoprecipitated HA-LIMK1 with no addition of immunoprecipitated SecPH was taken as 1.0. Each value represents the mean ± SE of two independent experiments. Immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining.</p

Schematic representation of our findings: A molecular connection between neurofibromin and the Rho/ROCK/LIMK2/cofilin pathway.

<p><i>A.</i> Upon Rho activation <i>via</i> binding to its RBD (Rho Binding domain), ROCK activates LIMK2 by phosphorylation at its Thr505. Activated LIMK2 will then phosphorylate cofilin on its Ser3, resulting in its inhibition. An invasive phenotype is then observed with accumulation of actin stress fibers. <i>B. SecPH, a new inhibitor of this Rho/ROCK/LIMK2/cofilin pathway</i>. By interacting with LIMK2, SecPH prevents ROCK activation of LIMK2. Our data raises two possible hypotheses for this inhibition of ROCK activation of LIMK2 by SecPH: (1) by steric hindrance, SecPH hides Thr505 of LIMK2 from ROCK accessibility, (2) the PH domain of SecPH substitutes to the PH domain of ROCK by inhibiting the kinase activity of ROCK; this inhibition would specifically occur when ROCK and SecPH are simultaneously bound to LIMK2.</p

SecPH inhibition of cofilin phosphorylation by LIMK2 requires ROCK activation of LIMK2.

<p><i>A. SecPH interacts with LIMK2 whatever its activation state.</i> Cells were cotransfected with SecPH and either LIMK2-WT, or LIMK2-TA or LIMK2-TEE. Lysates and anti-flag immunoprecipitates were subjected to immunoblotting. <i>B. Nf1 interacts with LIMK2 whatever its activation state.</i> Cells were transfected with either LIMK2-WT, or LIMK2-TA or LIMK2-TEE. Lysates and anti-HA immunoprecipitates were subjected to immunoblotting. <i>C. SecPH is unable to modulate cofilin phosphorylation by LIMK2-T505 mutants</i>. Cells were cotransfected with either LIMK2-WT, or LIMK2-TA or LIMK2-TEE and SecPH or its parental empty plasmid. Immunoprecipitated HA-LIMK2 and GST-cofilin were used in the kinase assay. Anti-HA immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining.</p

List of plasmids used in this study.

<p>List of plasmids used in this study.</p

SecPH affects ROCK kinase activity specifically with respect to LIMK2.

<p><i>A. MLC phosphorylation is not affected by SecPH.</i> Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone-0047283-g002" target="_blank">Figure 2B</a>, except 0.5 µg of MLC were added in the kinase reaction mixture. Each value represents the mean ± SE (standard error) of two independent experiments. B. <i>MLC phosphorylation by ROCK-1 is not affected by SecPH.</i> Cells were cotransfected with ROCK1 and SecPH or Galectin-3. Immunoprecipitated c-Myc-ROCK1 was used for the kinase assay in the presence of recombinant LIMK2 or MLC (0.5 µg each). The kinase activity on LIMK2/MLC of immunoprecipitated c-Myc-ROCK from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. Immunoprecipitates were also submitted to c-Myc-immunoblotting and lysates to flag-immunoblotting.</p

Interaction between LIMK2 and the SecPH domain of Nf1.

<p><i>A. Diagram of Nf1.</i> GRD (GAP related domain) responsible for the main known function of Nf1 is depicted as well as SecPH, the region used for the two-hybrid screening. <i>B. Interaction revealed by the two-hybrid screening.</i> L40 cells transformed with pBTM116-TBD were mated with Y187 cells transformed with the empty plasmid pACT2 (as a negative control) or pACT2-TFS1 (as a positive control, as demonstrated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone.0047283-Chautard1" target="_blank">[32]</a>). L40 cells transformed with pBTM116-SecPH were mated with Y187 cells transformed with pACT2-LIMK1 or pACT2-LIMK2. After mating on YPD, the resultant diploids were selected on a SD-LW medium. The interaction between the LexA fusion proteins encoded by the pACT2 plasmids and the Gal4 fusion proteins encoded by pBTM116 plasmids was tested by checking the growth of diploids on a SD-LWH media containing 3AT (1 mM) and their ability to cleave X-gal (1 mM) thereby attesting the production of β-galactosidase. <i>C. Interaction in HEK-293 transfected cells.</i> HEK-293 cells were cotransfected with either HA-LIMK2 or HA-LIMK1 and flag-SecPH or its parental empty plasmid (p3XFlag). Cell lysates and anti-flag immunoprecipitation eluates were analyzed by immunobloting. <i>D. Immunoprecipitated LIMK2 interacts with recombinant 6His-SecPH.</i> HEK-293 cells were transfected with HA-LIMK2 or its parental empty plasmid, pcDNA3. The corresponding cell lysates were immunoprecipitated with anti-HA beads. Beads were then incubated with 6His-SecPH in lysis buffer. Anti-HA immunoprecipitates were analyzed by immunobloting. <i>E. Transfected LIMK2 interacts with endogenous Nf1.</i> Cells were transfected with HA-LIMK2 or its parental empty plasmid, pcDNA3. Lysates and anti-HA immunoprecipitates were analyzed by immunobloting. <i>F. Endogenous LIMK2 interacts with endogenous Nf1</i> Anti-Nf1 immunoprecipitates from HEK-293 were analyzed by immunobloting. <i>G. Domains of LIMK2 involved in its interaction with SecPH</i>. Top. Schematic diagram of LIMK2 and its various fragments designed for this study. Bottom. Cells were cotransfected with SecPH and one of the domains of LIMK2. Lysates and anti-flag immunoprecipitates were analyzed by immunobloting.</p

Mechanism of SecPH inhibition of LIMK2 kinase activity.

<p><i>A. SecPH does not prevent LIMK2 from interacting with ROCK1</i>. Cells were cotransfected with HA-LIMK2, ROCK1 and SecPH or its empty parental plasmid. Lysates and anti-HA immunoprecipitates were subjected to immunoblotting. <i>B. SecPH affects LIMK2 phosphorylation.</i> Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone-0047283-g002" target="_blank">Figure 2B</a>. Statistical significance was determined relative to control using one-way ANOVA (*** p<0.0001). <i>C. Inhibition of LIMK2 cofilin phosphorylation by SecPH in the presence of ROCK1.</i> Cells were cotransfected with ROCK1, LIMK2 and SecPH or Galectin-3 (a non-specific control protein). Immunoprecipitated HA-LIMK2 and GST-cofilin were used for the kinase assay. The kinase activity on cofilin of immunoprecipitated HA-LIMK2 from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. The HA-immunoprecipitates were also submitted to immunoblotting and to coomassie blue staining. Lysates were also submitted to flag-immunoblotting. <i>D. SecPH affects LIMK2 T505 phosphorylation by ROCK1.</i> Cells were cotransfected with either LIMK2-TA or LIMK2-WT and SecPH or Galectin-3 (a non-specific control protein). Lysates were subjected to immunoblotting.</p

Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

International audienceMobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host