RNA stores tau reversibly in complex coacervates
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.
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 Cross-Linking ImmunoPrecipitation (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 S1 Data.