Journal: PLOS Biology

Article Title: A hormone-dependent tRNA half promotes cell cycle progression via destabilization of p21 mRNA

doi: 10.1371/journal.pbio.3003194

Figure Lengend Snippet: (A) Quantification of mature tRNA LysCUU and its 5′-half by RT-qPCR and TaqMan RT-qPCR, respectively, upon transfection of the indicated siRNAs. For all bar graphs in the present study, error bars indicate mean ± SD of triplicate measurements (* P < 0.05, ** P < 0.01, and *** P < 0.001; two-tailed t -Test). (B) The relative abundance of LNCaP cells af t er transfection of the indicated siRNAs. Abundance on the day of transfection was set at 1. Each dataset represents the average of three independent experiments with error bars showing the SD. (C) Representative images of the cells upon the siRNA transfection (Day 3). (D) The six most enriched GO terms upon KDs of tRNA halves, revealed using Panther enrichment analysis. No GO term with significant changes ( P < 0.001) was detected upon the KD of 3′-tRNA AspGUC half. (E) Cell cycle analyses using flow cytometry upon KDs of the indicated tRNA haves. The data underlying the graphs can be found in .

Article Snippet: Total cell lysate or cytosolic fraction of LNCaP cells was pre-cleared with Streptavidin Sepharose High Performance (GE Healthcare Life Sciences) in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM MgCl 2 , 0.5% NP-40, 0.1% Triton X-100, and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche).

Techniques: Quantitative RT-PCR, Transfection, Two Tailed Test, Flow Cytometry

Journal: PLOS Biology

Article Title: A hormone-dependent tRNA half promotes cell cycle progression via destabilization of p21 mRNA

doi: 10.1371/journal.pbio.3003194

Figure Lengend Snippet: (A) Nuclear, cytosolic, and mitochondrial fractions of LNCaP cells were subjected to RT-qPCR for the quantification of U6 snRNA, mitochondrial 12S rRNA, and 18S rRNA, as well as to TaqMan RT-qPCR for the quantification of 5′-tRNA halves. U6 snRNA, mitochondrial 12S rRNA, and 18S rRNA served as controls for nuclear, mitochondrial, and cytosolic RNA, respectively. (B) Proteins interacting with 5′-tRNA LysCUU half from pull-down experiments were developed by SDS–PAGE. Three prominent bands were subjected to mass-spec protein identification. The identified Top 5 proteins are shown. (C) The total protein lysate (Whole), nuclear, and cytosolic fractions of LNCaP cells were subjected to western blots. H2B and GAPDH were used as nuclear and cytosolic protein controls, respectively. (D) Cytosolic lysates from pull-down experiments using biotinylated control RNA or 5′-/3′-tRNA LysCUU half were subjected to western blot for YBX1. Input lysates before pull-down experiments were also analyzed as a control. (E, G) Total RNAs extracted from YBX1 KD (E) or OE (G) cells were subjected to RT-qPCR/TaqMan RT-qPCR for quantification of YBX1 mRNA, p21 mRNA, and 5′-tRNA LysCUU half. For all bar graphs in the present study, error bars indicate mean ± SD of triplicate measurements (* P < 0.05, ** P < 0.01, and *** P < 0.001; two-tailed t -Test). (F) Lysates from the YBX1 OE cells were subjected to western blots. (H) The relative abundance of the cells after transfection of the indicated siRNAs. To ensure consistency, the total amounts of siRNA were adjusted to be identical. The condition transfected only with control siRNA contained double the amount of the control siRNA, as the other two conditions contained two species of siRNAs. Abundance on the day of transfection was set at 1. Raw images of B, C, D, and F are located in S1 Raw images. The data underlying the graphs can be found in .

Article Snippet: Total cell lysate or cytosolic fraction of LNCaP cells was pre-cleared with Streptavidin Sepharose High Performance (GE Healthcare Life Sciences) in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM MgCl 2 , 0.5% NP-40, 0.1% Triton X-100, and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche).

Techniques: Quantitative RT-PCR, SDS Page, Mass Spectrometry, Western Blot, Control, Two Tailed Test, Transfection

Journal: PLOS Biology

Article Title: A hormone-dependent tRNA half promotes cell cycle progression via destabilization of p21 mRNA

doi: 10.1371/journal.pbio.3003194

Figure Lengend Snippet: (A) Absolute abundance of 5′-tRNA LysCUU half and p21 mRNA was quantified by TaqMan RT-qPCR and RT-qPCR, respectively, and calculated based on standard curves from quantification of various amounts of targeted RNAs. Averages of three independent experiments with SD values and the ratio of the abundance of 5′-tRNA LysCUU half versus p21 mRNA are shown. (B) UV-crosslinking and immunoprecipitation of YBX1, followed by western blot detection of the purified YBX1 (left) and TaqMan RT-qPCR/RT-qPCR detection of the 5′-tRNA LysCUU half and p21 mRNA (right). For all bar graphs in the present study, error bars indicate mean ± SD of triplicate measurements (* P < 0.05, ** P < 0.01, and *** P < 0.001; two-tailed t Test). (C) The positions and sequences of four LLs in p21 mRNA. Iden t ity rates to the sequences of the 5′-tRNA LysCUU half are shown. (D) Reporter constructs for luciferase assays to analyze the influence of LLs on mRNA stability. The CDS (with or without silent mutations in LL332) or the 3′-UTR (with or without LL588, LL1554, or LL1861) of p21 mRNA was attached to the downstream of Rluc mRNA. (E) Luciferase reporter assay. The constructs were transfected into LNCaP cells, followed by analysis of luciferase activity. (F) Biotinylated RNAs for pull-down experiments. Biotinylated p21 mRNA fragment of positions 578–638 (LL588-Bio: LL588 sequences with additional 10 nucleotides of 5′- and 3′-sequences) and biotinylated control RNA (Control-Bio: LL588 sequences in LL588-Bio were replaced with Rluc sequences) were used. (G) Pull-down experiments. The beads from the pull-down experiments using the indicated biotinylated RNAs were subjected to western blot for YBX1. Input lysates before pull-down experiments were also analyzed as a control. (H) Pull-down competition assay in which the beads from pull-down experiments using 5′-LysCUU-Bio or LL588-Bio were incubated with the competitor LL588 RNA or 5′-tRNA LysCUU half, respectively, followed by washing and western blot for YBX1 protein. Input lysates before pull-down experiments were also analyzed as a control. (I) Competition assay using DRaCALA. Recombinant YBX1 protein was incubated with either 32 P-labeled 5′-tRNA LysCUU half and LL588 or 32 P-labeled LL588 and 5′-tRNA LysCUU half. Unlabeled RNAs were used as competitors at concentrations of 100 μM or 200 μM. The bar graph represents the relative amounts of the indicated 32 P-IabeIed RNAs bound to YBX1 Raw images of B, G, H, and I are located in S1 Raw images. The data underlying the graphs can be found in .

Article Snippet: Total cell lysate or cytosolic fraction of LNCaP cells was pre-cleared with Streptavidin Sepharose High Performance (GE Healthcare Life Sciences) in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM MgCl 2 , 0.5% NP-40, 0.1% Triton X-100, and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche).

Techniques: Quantitative RT-PCR, Immunoprecipitation, Western Blot, Purification, Two Tailed Test, Construct, Luciferase, Reporter Assay, Transfection, Activity Assay, Control, Competitive Binding Assay, Incubation, Recombinant, Labeling