A new function for eukaryotic elongation factor 3: inhibition of stop codon readthrough in yeast (2023)

Cell Reports, Volume 15, Issue 8, 2016, pp. 1795-1808

InDrosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major cofactor. Previous analyzes indicate thisloqsMutants only partially impede miRNA processing, but the activity of smaller isoforms or maternally deposited loqs was not eliminated in these studies. We addressed this by creating a cell line fromloqs-null embryos and found that only about 40% of the miRNAs showed clear Loqs dependence. A genome-wide comparison of hairpin structure and Loqs dependency suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificially altering base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs-dependent. We propose that Loqs may play a role in aiding the de novo emergence of miRNA genes by facilitating the dicing of suboptimal hairpin substrates.

Cell, Volume 169, Issue 6, 2017, pp. 1051-1065.e18

During eukaryotic evolution, ribosomes have increased in size significantly, forming a surface-exposed ribosomal RNA (rRNA) envelope of unknown function that can provide an interface for as yet uncharacterized interacting proteins. To study such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories such as metabolism and cell cycle, as well as RNA and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we uncover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define their direct substrates. Furthermore, we show that the metabolic enzyme muscle pyruvate kinase (PKM) interacts with subpools of endoplasmic reticulum (ER)-associated ribosomes and exerts a non-canonical function as an RNA-binding protein in the translation of ER-determined mRNAs. Therefore, RAPs connect one of life's oldest molecular machines to diverse cellular processes, providing an additional layer of regulatory potential for protein expression.

Journal of Molecular Biology, Band 426, Ausgabe 1, 2014, S. 185-198

Membrane protein folding begins in the ribosome when the peptide is elongated. During this process, the nascent peptide navigates along a 100 Å tunnel from the peptidyltransferase center to the exit port. Proximal to the exit aperture is a “folding vestibule” that allows the nascent peptide to condense and explore the conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now show, using an accessibility assay and an improved intramolecular cross-linking assay, that the transmembrane helical hairpin (“paddle”) S3b-S4 of a voltage-gated potassium channel (Kv), a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Furthermore, this vestibular hairpin is consistent with a closed conformation of the Kv channel in the plasma membrane.

Journal of Biological Chemistry, Band 288, Ausgabe 47, 2013, S. 33697-33707

As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39, expose domains inside the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunneling proteins and nascent chains. As model proteins we used Dap2, which contains a hydrophobic signaling anchor (SA) segment, and the chimera Dap2α, in which the SA has been replaced by a hydrophilic segment with a propensity to form an α-helix. Using a newly developed FLAG exposure assay, we found that the nascent SA segment, but not the hydrophilic segment, assumed a stable α-helical structure within the tunnel when the C-terminal-most SA residue was extended by 14 residues from the Peptidyl transferase center was separated. Using UV crosslinking, antibodies specifically recognizing Rpl17 or Rpl39 and a His₆-tagged version of Rpl4, we found that all three yeast tunnel proteins contact the SA, while only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the exposed tunneling domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with residues of the nascent chain throughout the ribosomal tunnel.

Biochemie, Band 103, 2014, S. 89-100

Human ribonuclease inhibitor (RI), a cytoplasmic protein, is composed almost entirely of leucine-rich repeats. RI could suppress the activities of ribonuclease and angiogenin (ANG) by closely combining them. ANG is a potent inducer of blood vessel growth and has been implicated in tumor establishment, growth and metastasis. The ILK/PI3K/AKT signaling pathway also plays an important role in cell growth, cell cycle progression, tumor angiogenesis and cell apoptosis. Our previous experiments showed that RI could effectively inhibit part of tumor growth and metastasis. Our recent study showed that ILK siRNA inhibited growth and induced apoptosis in bladder cancer cells and increased RI expression, suggesting a correlation between RI and ILK. However, the exact molecular mechanism of RI in the anti-tumor and cross-talk of ANG and ILK signaling remains largely unknown. Here we examined the effects of RI upregulation on growth and apoptosis in murine melanoma cells via angiogenin and ILK/PI3K/AKT signaling pathways. We have shown that upregulation of RI apparently decreases ANG expression and activity. We also discovered that RI overexpression can remarkably inhibit cell proliferation, regulate cell cycle and induce apoptosis. Furthermore, upregulation of RI inhibited phosphorylation of the downstream ILK signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β) and reduced β-catenin expression in vivo and vitro. More importantly, RI significantly inhibited tumor growth and angiogenesis in tumor-bearing C57BL/6 mice. Taken together, our results suggest for the first time that angiogenin and ILK signaling play a central role in mediating the inhibitory effect of RI on melanoma cell growth. This study identifies that RI may be a useful molecular target for melanoma therapy.

Journal of Pharmaceutical Sciences, 2023

Loss of the phosphatase and tensin homologue (PTEN) deleted from chromosome 10 is commonly observed in a variety of human cancers and appears to be an ideal target for synthetic lethality-based treatment. In this study, the synthetic lethal interaction between PTEN loss and poly[ADP-ribose] polymerase 1 (PARP1) gene silencing in human triple-negative breast cancer cells (PTEN-null MDA-MB-468 and PTEN-positive) examines MDA-MB-231 cells). Previously developed by us, polycationic liposomes were used to deliver the small interfering ribonucleic acid (siRNA) targeting PARP1 (siPARP1) into cancer cells. The silencing of the PARP1 gene exerted a cytocidal effect on the MDA-MB-468 cells, but had no effect on the MDA-MB-231 cells and the human umbilical vein endothelial cells used as normal cells. The simultaneous knockdown of PARP1 and PTEN in the MDA-MB-231 cells led to a significant inhibition of cell growth. The data suggest that the effects of PARP1 knockdown on cells were dependent on PTEN status. A significant increase in DNA breaks and the extent of apoptosis, possibly due to DNA repair failure, was observed after PARP1 knockdown in the MDA-MB-468 cells compared to the case in the MDA-MB-231 cells . Our results suggest that the synthetic lethal approach via PARP1 gene silencing holds promise for treating patients with PTEN-null breast cancer.