Presentation on theme: "Supp Figure S1, Yang et al.Supp Figure S2, Yang et al. Supp Figure S1. Calorimetric analysis of La(1-194) and LARP4(111-303) interactions with single stranded."— Presentation transcript:
Supp Figure S1, Yang et al.Supp Figure S2, Yang et al. Supp Figure S1. Calorimetric analysis of La(1-194) and LARP4(111-303) interactions with single stranded RNA sequences. Raw titration data showing the thermal effect of: injecting U(15) into a calorimetric cell containing La(1-194) (A) or LARP4(111-303) (D), injecting A(10) into La(1-194) (B) or LARP4(111-303) (E) and injecting A(15) into La(1-194) (C) or LARP4(111- 303) (F). (G-L) The normalised heat for the titrations shown in A-F respectively was obtained by integrating the raw data and subtracting the heat of ligand dilution. The heat effect reveals null to weak interactions in B, C, D and E and a strong association in A and F. The grey lines in G and L represent the best fit derived by a non-linear least-squares procedure based on an independent binding sites model. Supp Figure S2. The ITC titration experiment of LARP4 with the A(15) (raw data and fitted curve, panel B and C respectively) is reported together with the A(15) dilution control experiment (panel A), in which the RNA (at the same concentration, same temperature, same buffer etc.) was titrated into the calorimetric cell containing the buffer alone. Here, in the control experiment all the peaks of the thermogram have an equivalent height, reporting exclusively the heat of dilution of A(15) (same amount, for each injection) in the buffer. The amount of heat corresponding to the control dilution experiment of A(15) (in panel A) matches the one recorded at the end of the binding experiment with the protein (Panel B), indicating that, in presence of the protein, the last peaks recorded in the titration result exclusively from the dilution of the oligonucleotide.
PAM2w La motif 2 1 1 3 2 4 L2L3L4 Supp Figure S3, Yang et al. * Multiple sequence alignmwent of LARP4 and LARP5 proteins from several species. The human LARP4 sequence is followed by human LARP5, separated by a thin horizontal line. The vPAM2 and La motifs are indicated. The RRM is indicated by its secondary structure features as predicted for human LARP4 by the Protein Structure Prediction Meta Server (Kajan and Rychlewski. 2007. BMC Bioinformatics. 8:304). The Q->T sequence difference (iasterisk) is a key residue involved in base-specific RNA recognition observed in La-RNA crystals (Teplova et al. 2006. Mol Cell 21:75-85, Kotik-Kogan et al. 2008. Structure 16:852-62}.
110 9.0 115 8.0 120 7.0 105 1 H (ppm) 15 N (ppm) 110 A)B) 9.08.0 1 H (ppm) 105 15 N (ppm) G579 T576 G583 G563 Figure S4. 15 N- 1 H NMR correlation spectra of the 15 N-labeled MLLE domain from PABPC1 titrated with increasing amounts of the LARP4 PAM2w peptide. A) Overlays of five spectra: red, no addition; green, 0.07 mM peptide; cyan, 0.11 mM peptide; purple, 0.15 mM peptide; violet, 0.26 mM peptide. The starting protein concentration was 0.2 mM. B) Expansion of the upfield region showing peaks with residue numbers shifting in fast and intermediate exchange. Spectra were acquired on a Bruker Avance 600 MHz spectrometer in 50 mM NaH 2 PO 4, 100 mM NaCl, pH 7.0 at 25 °C. Supp Figure S4, Yang et al.
Supp Figure S6, Yang et al. Expression of F-LARP4 increases mRNA half-life. Shown are the individual decay curves for each of the clustered decay curves in Fig. 9.
KL MNO JI P Hoechstanti-FLAG anti-TIA-1 merge Supp Figure S7, Yang et al. F-LARP4 and endogenous LARP4 localize to stress granules. I-P, Flag-LARP4; Q-X, endogenous LARP4. I-P) After transfection with F-LARP4, cells were mock treated (Fig. 3I-L) or treated with arsenite (Fig. 3 M-P) which is used widely to induce stress granules and examined with anti-FLAG (green) and anti-TIA-1 (red) antibody. LARP4 distribution was homogeneously cytoplasmic in the mock treated cells (Fig. 3J) whereas a significant fraction became localized in punctate foci after arsenite (Fig. 3N). Stress granule marker TIA-1 also organized into distinct loci after arsenite (Fig. 3K vs O). Image merging revealed superimposed LARP4 foci and TIA-1 foci (Fig. 3P). The data are consistent with the idea that F-LARP4 is associated with mRNPs engaged for translation initiation that assemble into stress granules after exposure to arsenite. Q-X) Cells were mock treated (Fig. 3Q- T) or treated with arsenite (Fig. 3 U-X) and examined with anti-LARP4 (green) and anti-FMRP (red) antibody. LARP4 distribution was mostly cytoplasmic in the mock treated cells (Fig. 3R) whereas a significant fraction became localized in more concentrated foci after arsenite (Fig. 3V). Stress granule marker FMRP (see Schaffler et al. 2010) organized into the same loci after arsenite (Fig. 3S vs W). Image merging revealed superimposed LARP4 foci and FMRP foci (Fig. 3X). The data are consistent with the idea that F-LARP4 is associated with mRNPs engaged for translation initiation that assemble into stress granules after exposure to arsenite. ST UVW RQ X Hoechstanti-LARP4 anti-FMRP merge
Supp Figure S8, Yang et al. siRNA-mediated knockdown of LARP4 reduces FAIM mRNA half-life. Three graphs show decay of FAIM mRNA corrected for loading by the MRP snRNA, GAPDH mRNA and 18S rRNA.