The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) com5116CNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)202144/2011-9We thank J Warner, A van Hoof, R Kopito, O Brandman, and S Lindquist for reagents. EBT gratefully acknowledges the Brazilian Council for Scientific and Technological Development (CNPq) for a Postdoctoral Fellowship. MK was supported by the Hartmut Hoffma

The intronic region of Fbxl12 functions as an alternative promoter regulated by UV irradiation

AbstractThe ubiquitin ligases, SCF complexes, consist of Cul1, Skp1, Rbx1 and the substrate recognition components F-box proteins. Previous studies have reported that one of these F-box proteins, Fbl12, which is produced by Fbxl12 gene, regulates both cell cycle and differentiation. In this paper, we show that the intronic region of Fbxl12 gene acts as an alternative promoter and induces expression of a short form of Fbl12 that lacks F-box domain (Fbl12ΔF). We also found that UV irradiation increases Fbl12ΔF mRNA in cells. Finally, Fbl12ΔF may promote the subcellular localization of Fbl12 from nucleus to cytoplasm through their binding. Our data provide the possibility that Fbl12ΔF induced by alternative promoter controls the SCFFbl12 activity in response to UV stimulation

Myeloma Overexpressed 2 (Myeov2) Regulates L11 Subnuclear Localization through Nedd8 Modification

<div><p>Nucleolus is a dynamic structure that controls biogenesis of ribosomal RNA and senses cellular stresses. Nucleolus contains a number of proteins including ribosomal proteins that conduct cellular stresses to downstream signaling such as p53 pathway. Recently, it has been reported that modification by a ubiquitin-like molecule, Nedd8, regulates subnuclear localization of ribosomal protein L11. Most of L11 is normally localized and neddylated in nucleolus. However, cellular stress triggers deneddylation and redistribution of L11, and subsequent activation of p53. Although Nedd8 modification is thought to be important for L11 localization, the mechanism of how neddylation of L11 is regulated remains largely unknown. Here, we show that Myeloma overexpressed 2 (Myeov2) controls L11 localization through down-regulation of Nedd8 modification. Expression of Myeov2 reduced neddylation of proteins including L11. We also found that Myeov2 associates with L11 and withholds L11 in nucleoplasm. Although Myeov2 interacted with a Nedd8 deconjugation enzyme COP9 signalosome, L11 deneddylation was mediated by another deneddylase Nedp1, independently of Myeov2. Finally, p53 transcriptional activity is upregulated by Myeov2 expression. These data demonstrate that Myeov2 hampers L11 neddylation through their interactions and confines L11 to nucleoplasm to modulate nucleolar integrity. Our findings provide a novel link between oncogenic stress and p53 pathway and may shed light on the protective mechanism against cancer.</p></div

Model for the role of Myeov2 in nucleus.

<p>(<b>A</b>) Most of L11 is neddylated and localized in nucleolus. In response to nucleolar stress, L11 is deneddylated and relocates to nucleoplasm, leading to activation of p53. (<b>B</b>) Myeov2 blocks L11 neddylation by their interaction and suppresses translocation of L11 into nucleolus, and make cells sensitive to stresses.</p

Expression of Myeov2 maintains L11 in a lower neddylation level in cells.

<p>(<b>A</b>) HEK293 cells were transfected with HA-Nedd8 and either Flag-Myeov2 or Flag-Myeov2ΔFD. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (<b>B</b>) HEK293T cells were transfected with either Flag-Myeov2 or Flag- Myeov2ΔFD and then treated with 10 µM MG132 for 1 hour prior to harvest. Cell lysates were subjected to immunoblot analyses. Arrowhead indicates putative Myeov2 target protein. (<b>C, D and E</b>) HEK293T cells were transfected as indicated. Neddylated proteins were precipitated from denatured cell lysates using Talon metal affinity resin and immunoblotted with indicated antibodies. Arrowheads indicate neddylated L11.</p

Myeov2 alters L11 subnuclear localization without nucleolar disruption.

<p>(<b>A</b>) HeLa cells were transfected with Myc-L11, together with either Flag-Myeov2 or Flag- Myeov2ΔFD plasmids, and subjected to immunocytochemistry. Arrows and arrowheads indicate Myeov2-expressing and control cells, respectively. Scale bar, 20 µm. (<b>B</b>) Quantification of the data in (A). Subnuclear localization of Myc-L11 in Flag-Myeov2-positive cells was examined. Cells that express L11 in nucleous only (yellow), both in nucleolus and nucleoplasm (blue) and nucleoplasm only (pink), were counted. N denotes the number of cells counted. (<b>C</b>) Co-immunoprecipitation of Flag-Myeov2 or Flag-Myeov2ΔFD with HA-L11 in HEK293T cells. Cell lysates were immunprecipiated with anti-Flag antibodies and analyzed using both Flag and HA antibodies. (<b>D</b>) HeLa cells transfected with EGFP-NPM and HA-Myeov2 were treated with 10 nM ActD for indicated time, and subjected to immunocytochemistry. Expression of Myeov2 did not induce nucleolar disruption in the absence of ActD (top panels). Nuclear disruption was observed in Myeov2-expressing cells at 1 hr of ActD treatment (middle panels). 4 hrs of ActD treatment resulted in nucleolar disruption irrespective of Myeov2 expression (lower panels). Scale bar, 20 µm.</p