Journal: RNA

Article Title: Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

doi: 10.1261/rna.2152710

Figure Lengend Snippet: Analysis of MS2BP/MS2 binding by NIR-labeled RNA probes. (A) NIR-labeled UTPs (Jena Bioscience) were used for in vitro transcription. Dyes (Atto680 or DY776) were covalently bound to UTP (C-5 of pyrimidine ring) by a flexible aminoallyl linker. (B) Analysis of in vitro transcribed MS2 labeled with UTP-Atto680 (upper panel) or UTP-DY776 (lower panel). Indicated amounts of labeled RNA probes (fmol) were analyzed by infrared scanning of 4%–12% native TBE gels using a LI-COR Odyssey scanner. Used scanning intensities (maximum of 10) are indicated (right). (C) Filter binding analyses of the MS2BP/MS2-Atto680 association. RNA spotted on nitrocellulose filters (Input: 100%, 15 fmol) or immobilized on filters upon binding to indicated amounts of MS2BP was analyzed by infrared scanning. (D) The percentage of MS2-Atto680 RNA bound by indicated protein amounts was determined by input-normalized fluorescence intensities (C). The Hill equation was used for fitting (solid black line) of data up to 100 nM MS2BP and KD calculation. (Dotted line) Increased binding at protein concentrations >100 nM. (E) EMSA analysis of the MS2BP/MS2–Atto680 association. Complex formation of indicated amounts of MS2BP and MS2-Atto680 (15 fmol) was analyzed by EMSA in native 4%–12% TBE gels and infrared scanning. (fP) Free probe, (CI) Complex 1, (CII) Complex 2. (F) The percentage of MS2-Atto680 RNA bound by indicated MS2BP amounts was determined by input-normalized (I) fluorescence intensities (E) observed for CI (filled circle, solid line), CII (open circle, dotted line), or total binding (CI+CII: triangle, dashed line). The Hill equation was used for fitting of total binding (solid thick black line) using data up to 1 μM MS2BP. Error bars indicate SD from three independent analyses.

Article Snippet: For NIR dye labeling, in vitro transcription reactions (0.5 mM rATP, rCTP, and rGTP) were supplemented with 12.5 μM rUTP and 20 μM Atto680- or DY776-UTP (final concentration; Jena Bioscience).

Techniques: Binding Assay, Labeling, In Vitro, Fluorescence

Journal: RNA

Article Title: Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

doi: 10.1261/rna.2152710

Figure Lengend Snippet: Analysis of distinct protein–RNA complexes in one sample by NIR probes. (A) UV crosslinking of MS2-Atto680 (200 fmol) to MBP (3 μg) or indicated amounts of MS2BP after RNase A treatment and SDS-PAGE. Complex formation was analyzed by infrared scanning of the SDS-gel (A) followed by Western blotting with anti-MBP (B). (4×MS2BP) Tetramer, (2×MS2BP) dimer, (MS2BP) monomer, (MBP) maltose binding protein. (C) Competition of the MS2BP/MS2 association analyzed by EMSA. Binding of MS2-Atto680 (15 fmol) to MS2BP (80 nM) was competed by a 50-fold molar excess of unlabeled MS2 (lane 3) or MS2mut (lane 4). The percentage of bound probe (bottom panel) was determined as described in Figure 1. Sequence and stem–loop structure of used probes are indicated in Supplemental Figure 1A. (D) EMSA analysis of the MS2BP/MS2 and GST-λ/B-box association in one sample. Complex formation of MS2BP and/or GST-λ with MS2-DY776 (green, 25 fmol) and/or B-box-Atto680 (red, 15 fmol) was analyzed by EMSA. Note that both NIR RNAs were present in all samples. (Lane 1) Buffer control, (lane 2) MS2BP (80 nM), (lane 3) GST-λ (500 nM), (lane 4) MS2BP (80 nM) and GST-λ (500 nM). Free NIR probes and protein–RNA complexes are indicated. For color-separated scans, see Supplemental Figure 2C.

Article Snippet: For NIR dye labeling, in vitro transcription reactions (0.5 mM rATP, rCTP, and rGTP) were supplemented with 12.5 μM rUTP and 20 μM Atto680- or DY776-UTP (final concentration; Jena Bioscience).

Techniques: SDS Page, SDS-Gel, Western Blot, Binding Assay, Sequencing

Journal: RNA

Article Title: Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

doi: 10.1261/rna.2152710

Figure Lengend Snippet: ZBP1 associates with Y1 and Y3 RNAs. (A) Filter binding analysis of ZBP1 and Y-RNAs. Filter binding was performed as described in Figure 1, C and D. For fitting, the Hill equation was used, assuming RNA-dependent dimerization of ZBP1 (Nielsen et al. 2004; Chao et al. 2010). (B) Association of Flag-ZBP1 with Y3 in 293 cells. Flag-tagged ZBP1 or PKM2 were immunopurified from transiently transfected 293 cells as confirmed by Western blotting with anti Flag-M2 (upper panel, Western). Copurification of Y-RNAs or TRNAG1 was analyzed by semiquantitative RT-PCR using 26 amplification cycles for Y4, Y5, and TRNAG1 or 30 amplification cycles for Y3 (lower panels, RT-PCR). PCR analyses of IP samples without preceding reverse transcription (−RT) served as controls. (C) Enrichment of indicated RNAs upon ZBP1 or PKM2 immunopurification was determined by quantitative RT-PCR using the ΔCt-method and normalization to input samples. (D) Binding of ZBP1 to exogenous Y-RNAs in 293 cell lysates. Lysates were supplemented with Atto680-labeled Y-RNAs (500 fmol) and recombinant ZBP1 (500 nM) before UV crosslinking as indicated (X-Link). Complex formation was analyzed by infrared scanning of SDS gels and Western blotting with anti-ZBP1. (E) Y3 competes for binding of ZBP1 to the ACTB-3′UTR in EMSA studies. Binding of ZBP1 (120 nM) to the ACTB-3′UTR(1–100 nt) (50 fmol) was competed by indicated molar excess of unlabeled Y-RNAs. Free probe (fP) and the ZBP1/ACTB(100 nt) complex are indicated. Error bars indicate SD from three independent experiments.

Article Snippet: For NIR dye labeling, in vitro transcription reactions (0.5 mM rATP, rCTP, and rGTP) were supplemented with 12.5 μM rUTP and 20 μM Atto680- or DY776-UTP (final concentration; Jena Bioscience).

Techniques: Binding Assay, Transfection, Western Blot, Copurification, Reverse Transcription Polymerase Chain Reaction, Amplification, Immu-Puri, Quantitative RT-PCR, Labeling, Recombinant

Journal: RNA

Article Title: Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

doi: 10.1261/rna.2152710

Figure Lengend Snippet: Formation of a trimeric ZBP1-La-Y3 complex. (A,B) Binding of ZBP1 and/or La protein to DY776-labeled Y3 (25 fmol) and/or Atto680-labeled Y5 (625 fmol) was analyzed by EMSA as indicated. Specific protein–RNA complexes are indicated. (B) Color-separated scans for DY776 (800 nm) or Atto680 (700 nm). Note that the supershifted complex comprising ZBP1, La, and Y3 is dissociated in the presence of Y5. (C) Schematic of complexes observed in A. ZBP1 and La form a trimeric complex with Y3. La is dissociated in the presence of excess Y5. This results in the formation of two complexes of similar migration in EMSA: ZBP1/Y3 and La/Y5.

Article Snippet: For NIR dye labeling, in vitro transcription reactions (0.5 mM rATP, rCTP, and rGTP) were supplemented with 12.5 μM rUTP and 20 μM Atto680- or DY776-UTP (final concentration; Jena Bioscience).

Techniques: Binding Assay, Labeling, Migration