Polyu

Dendritic cells (DC) were prepared from C57BL/6 bone marrows as previously described (33 (link)). Briefly, bone marrow cells were flushed with ice-cold PBS and erythrocytes lysed using ACK buffer (8.7 g/L NH₄Cl, 1g/L KHCO₃, 0.05 mM EDTA, pH 7.3). Cells were cultured in DMEM 20% heat-inactivated FBS (Gibco), 1mM sodium piruvate, 2 mM glutamine and antibiotics (1 U/ml penicillin, 1 μg/ml streptomycin) (Invitrogen), in presence of 20 ng/mL mrGM-CSF and 10 ng/mL mrIL-4 (ImmunoTools). Cells were cultured for 5 days, with addition of GM+IL-4 every 2 days. Immature DCs were pulsed during 5 h with TRAMP-C1 lysates (4 freeze/thawing steps), and then matured with 2 ng/mL CpG-1826 (phosphorothioate 5'-TCCATGACGTTCCTGACGTT-3', IDT) and 20 μg/mL Poly-U (Sigma-Aldrich) during an overnight culture. To obtain experienced anti-tumor lymphocytes, wild type or Lgals1-/- mice were s.c. immunized with tumor lysate-pulsed dendritic cells. At day 7 pot-immunization, lymphocytes obtained from axillary, brachial and inguinal lymph nodes were used for immune reconstitution of nude mice.

Total RNA was isolated from HeLa cells using the RNeasy Mini Kit (QIAGEN). mRNA was isolated from total RNA using the DynabeadsTM mRNA purification Kit (ThermoFisher). The tangled total RNA was prepared according to published work [20 (link)]. Poly(A) (Sigma-Aldrich, Cat#10108626001), Poly(C) (Sigma-Aldrich, Cat#P4903), Poly(G) (Sigma-Aldrich, Cat#P4404), Poly(U) (Sigma-Aldrich, Cat#P9528), and Ribosomal RNA (Bioworld, Cat#11020001-2) were all purchased. The 5’UTR-KpnB1-nanoLUC mRNA was transcribed with an mMESSAGE T7 kit (Invitrogen).

DHX36 was incubated in K-Res buffer with 0.01 mM ATP spiked with 50 nCi [γ-³²P]-ATP in the presence of either poly(U) (100 ng·μl⁻¹, Sigma) or 100 nM Z33 G4 and 5 μM ligand. At indicated times, reactions were stopped by adding EDTA (0.5 M, 1 μl, pH 8.0) to a 5 μl aliquot of reaction. ATP hydrolysis was quantified by CEL PEI/UV254 TLC plates (Polygram) in 0.8 M acetic acid/0.8 M LiCl aqueous running solution after washing in methanol. TLC plates were air dried. Imaging was as for PAGE. Hydrolysis data were fit with one-phase exponential decay. One-way ANOVA was used to assess the significance of ATP hydrolysis data.

The tau protein was incubated at 20 μM in 384-well low-volume
microplate with 20 μM ThT. The RNA polyC (Sigma P4903), polyA
(Roche 10108626001) or polyU(Sigma P9528) were added at 200 μM.
Heparin (Sigma H6279) was added at a concentration of 5 μM in
supplementary experiments (Figure S11).
The working volume was 20 μL. The fluorescence was bottom read
in a BMG fluoroStar Omega instrument with excitation and emission
wavelengths of 440 and 480 nm, respectively. Each condition was prepared
independently in three or two different wells.
ThT kinetic curves
were first normalized between 0 and 1. They were then fitted using
the curve_fit python function with the following
equation: where F represents the final
fluorescent intensity, k represents the growth rate,
and t_1/2 represents the aggregation halftime.
Each replicate was fitted independently. The presented error bars
on the aggregation halftimes represent the standard deviation over
the output t_1/2 obtained from the fit
of each replicate. The fitting functions are shown in Figure S3. tau187-WT signal was normalized with
the maximum intensity of tau187-P301L for visualization in Figure 2A, since it did not
increase.

The nonspecific RNA binding ability of DRG1 proteins was assayed in vitro as described^{7 (link),69 (link)}. Briefly, 0.5 mg of proteins were incubated in 100 µL of cold reaction buffer (10 mM HEPES–NaOH (pH 7.4), 100 mM NaCl, 2 mM MgCl₂, 0.1% Triton X-100, 3 mM DTT) with a free poly(U) (Sigma-Aldrich) at concentrations of 0, 0.1 and 1 mg/mL. The reactions were incubated for 30 min at 4 °C in the rotator. Subsequently, 10 µL of 50% poly(U)-agarose (Sigma Aldrich) in binding buffer was added to each reaction mixture and incubated for 30 min at 4 °C. The beads were washed six times in binding buffer following centrifugation for 1 min at 4 °C. Finally, proteins bound to the poly(U)-agarose were eluted by adding 10 µL of 4 × SDS sample buffer and boiled at 95 °C for 6 min. Samples were loaded onto a 12% SDS-PAGE gel and visualized by Coomassie brilliant blue.