Tau tRNA binding by gel shift assay and isothermal titration calorimetry (ITC).

<p>(A) Direct titration experiment shows 4R2N tau induces a mobility shift in tRNA^Lys. To determine the K_d value, direct titration experiment was done, which requires trace RNA concentrations to meet the assumption used in the equation that derives the K_d value. The assumption is that the total protein concentration is approximately the free protein concentration at equilibrium, and that protein binding to RNA is negligible. In the direct titration experiments 26 nM, tRNA was used and protein concentration spans from 20 nM to 2 μM. (B) The fraction of bound tRNA by 4R2N tau plotted as a function of the monomeric tau concentration and fit to the Hill equation, y = 1 / [1 + (K_d / x)ⁿ]. (C) Yeast tRNA was titrated into solutions of 4R2N tau in an ITC experiment. The top panel shows the raw incremental-titration. The area under each peak is integrated and plotted against the tRNA:tau molar ratio and fitted to an independent binding model (the bottom panel), as discussed in Materials and methods. (B-C) SEM is reported from <i>n</i> = 3. (D) The stoichiometric binding experiments were performed by varying the tau:RNA molar ratio at a constant 2.6 μM concentration of tRNA, which approximates the saturation concentration (more than 5 times the K_d of 460 nM). The representative data of 3 independent experiments are shown. (E) Fraction of bound tRNA from the different bands in (D) is plotted over the molar tau:tRNA ratio. (F) Fraction of bound tau plotted as a function of tau:tRNA molar ratios and compared to the theoretical saturation binding curves (dotted lines) with protein:RNA molar stoichiometries of 1:1 to 6:1. The theoretical curves serve the purpose showing that multiple tau molecules bind tRNA with increasing tau concentrations, while the model is not meant to fit the data, given that multiple populations with different tau:tRNA ratios will coexist. The numerical data used in (B), (C), (E), and (F) are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

tRNA transfection accumulates sarkosyl insoluble tau in human-induced pluripotent stem cell (hiPSC) derived neurons.

<p>(A) Representative western blot of neurons harboring a P301L <i>tau</i> mutation transfected with tRNA, but not cells transfected in the absence of nucleic acids (Mock) present evidence for accumulation of sarkosyl insoluble tau (Ins.) as seen in the intense band in lane 9 labeled with PHF-1 antibody. Addition of tRNA to the lysis buffer is insufficient to increase the tau present in the insoluble fractions (compare Mock + tRNA, lane 6 to tRNA, lane 9). (B-C) Quantification of PHF-1 tau and β-actin level for both neurons harboring a P301L tau mutation (B) and neurons expressing wild type tau (C) are shown. Error bar represents standard error of the mean. * <i>p</i> < 0.05, *** <i>p</i> < 0.001, <i>n</i> > 3. The numerical data used in B and C are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

Enrichment of tRNA in PhotoActivatable Ribonucleoside-enhanced Individual-nucleotide resolution UV Cross-Linking ImmunoPrecipitation and high-throughput sequencing (PAR-iCLIP) data.

<p>(A) <u>C</u>ross-<u>L</u>inking <u>I</u>mmunoPrecipitation (CLIP) cDNA reads from tau expressed in human-induced pluripotent stem cell (hiPSC)-derived neurons (neuron CLIP) and from tau expressed in human embryonic kidney (HEK) cells (HEK cell CLIP) that aligned to the chr15.tRNA4-ArgTCG tRNA were found in all CLIP samples, and demonstrated a similar pattern of crosslinking. (B) Analysis of cross-linked sites along tRNA secondary structure demonstrates the anticodon preference, in which the anticodon (colored red) region is designated as position 1–3 for alignment purpose. The colored illustration of tRNA secondary structure is displayed as an inset, and below the <i>x</i> axis in one dimension. The numerical data used in (B) are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

Tau PhotoActivatable Ribonucleoside-enhanced Individual-nucleotide resolution UV Cross-Linking ImmunoPrecipitation and high-throughput sequencing (PAR-iCLIP) in tau-expressing human embryonic kidney cells (HEK) and human-induced pluripotent stem cell (hiPSC) -derived neurons.

<p>Phosphor images in the blue frames (A-C) show ³²P-labelled RNA crosslinked to tau protein in HEK cells expressing tau (A-B) and in hiPSC-derived neurons with endogenous tau (C). PAR-iCLIP in HEK cells expressing wild-type tau (A, lane 2) or tau P301L-CFP (B, lane 2; nota bene: the fused cyan fluorescent protein [CFP] retards the migration of tau). (C) PAR-iCLIP of endogenous wild-type tau in hiPSC-derived neurons (C, lane 2). The antibodies anti-tau HJ 8.5, anti-green fluorescence protein (GFP) and anti- Cyclin–dependent kinase 5 (CDK5) were used for protein precipitation. No RNase was added, unless specified. PAR-iCLIP with GFP as a control (A-B, lane 1), with CDK5 as a control (C, lane 3) and no UV control (C, lane 1). A small RNA signal was visible in the absence of cross-linking (no UV control) in hiPSC-derived neurons (C, lane 1), suggesting a small portion of RNA may associate with tau in vitro after cell lysis. The RNA-protein complexes from <u>C</u>ross-<u>L</u>inking <u>I</u>mmuno<u>P</u>recipitation (CLIP) marked by rectangles were cut from the blot for DNA library preparation. Note that 2 regions of GFP and CDK5 were cut out as sequencing controls in which the lower molecular weight (MW) band corresponds to GFP or CDK5. (D-E) Percentage of tau-CLIP reads that are mapped to 8 human genome regions in HEK cells (D) and hiPSC-derived neurons (E). (F-G) Enrichment of tRNA in tau-CLIP of HEK cells (F) and hiPSC-derived neurons (G) as discussed in text. The numerical data used in (D-G) are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

Tau in condensed droplets assumes solution state properties.

<p>(A-C) Continuous wave electron paramagnetic resonance (cw EPR) spectra obtained at room temperature of 500 μM Δtau187-SL in droplets formed with 1.5 mg/ml poly(A) RNA (blue in A) and 1.5 mg/ml tRNA (green in B) is unaltered from solution before adding RNA (red in A and B). Cw EPR line shape upon adding 125 μM heparin (black in C) show dramatic line broadening (compared to red in C). Double electron-electron resonance (DEER) of Δtau187-SL₂ in droplets formed with 1.5 mg/ml poly(A) RNA (blue in D) and 1.5 mg/ml tRNA (green in E), as well as upon incubation with 137.5 μM heparin (black in F) compared to Δtau187-SL₂ in solution (red in D-F). The numerical data are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

Tau and RNA forms droplet in vitro.

<p>(A) Bright field snapshots of droplets from 400 μM Δtau187 and 800 μg/ml poly(A) showing 2 droplets seamlessly fusing (highlighted with red circle). (B) Confocal microscopy image of Δtau187 labeled with Alexa-488 of the droplets from the same sample as in (A). 50 μM Alexa-488 labeled Δtau187 mixed with 350 μM MTSL labeled Δtau187 of droplets formed immediately after adding 800 μg/ml poly(A) RNA. Both (A) and (B) were sampled at room temperature and without added NaCl.</p

The G55R mutation increases the ability of 4R tau but not 3R tau to nucleate microtubule assembly.

<p>(A) Microtubule assembly in reactions containing a 1∶30 tau:tubulin dimer molar ratio were assayed by light scattering as a function of time. (B) Co-sedimentation assays demonstrate that the G55R mutation does not affect the ability of tau to assemble MT mass at steady-state, nor does it affect the ability of tau to bind to microtubules. Statistical significance was determined by comparing each mutant to its corresponding WT using two-tailed t-tests. Data in both panels represent the mean ± SEM from three independent experiments.</p

Schematic map of the six CNS tau isoforms.

<p>Exons 2 (E2), 3 (E3) and 10 (E10) are alternatively spliced to generate all six possible combinations. Arrowheads denote the position of the G55R mutation, present in four of the six isoforms. R1, R2, R3 and R4 denote the four imperfect repeats in the MT binding region.</p

A. The family tree of the affected family shows the pattern of inheritance.

<p>The proband is the black oval on the left side of the figure (II:1), marked with an arrow. Tau haplotypes of sequenced individuals are also noted. “aoo” corresponds to age of onset; “aod” corresponds to age of death; black filling indicates persons possessing the G55R mutation; gray filling corresponds to diagnosed dementia of unknown origin (presumed to be G55R but inadequate medical records exist). Proband's son III:1 (from first marriage) is 36 years old and a carrier of G55R. Proband's second son III:2 (from second marriage) is 31 and also a G55R carrier. The other two sons (III:3 and III:4; from the second marriage) are not G55R carriers and are 29 and 28 years old. <b>B. The tau sequence in the region of the G55R mutation is extremely highly conserved across species lines.</b> The glycine at position 55 is completely conserved in seven species ranging from humans to lizards. Color coding emphasizes conserved nature of acidic (red), basic (blue), hydrophilic/polar (orange), hydrophobic (green) and proline (peach) positions.</p